Structural nucleic acid guided chemical synthesis

ABSTRACT

Disclosed is a composition comprising the nucleic acid and a chemical compound, said composition forming a star structure defining 3 or more stems extending from a reaction center. The stems are formed by a nucleic acid duplex and the chemical compound has been formed in the reaction center as the reaction product of 3 or more chemical groups. The advantage of the composition is that a close proximity is provided between the chemical groups in the reaction center, thereby promoting a reaction. The invention also relates to a method for preparation of the composition. The advantage of the method is that it does not require the pre-synthesis of a large number of templates and that it is not dependent upon codon/anti-codon recognition for an encoded molecule to be formed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This a divisional of U.S. patent application Ser. No. 11/577,649 (nowU.S. Pat. No. 8,202,823), which is a 371 National Stage Entry ofPCT/DK2005/000714, filed Nov. 8, 2005, which claims benefit from U.S.Provisional Application Nos. 60/687,849, filed Jun. 7, 2005, and60/725,347, filed Oct. 11, 2005. The entire disclosures of the priorapplications are considered part of the disclosure of the accompanyingapplication and are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for in vitro DNAdisplay technology, allowing display of variety of molecules, such asnon-natural polymers and small molecules. Advantages of such methods arethat combinatorial libraries can be constructed and subjected to roundsof selections for desired activities, amplification and diversification,thus allowing molecular evolution.

BACKGROUND

Display technologies have been developed to combine information storageand amplification capabilities of nucleic acids with the functionalactivities of other compound. Display technologies rely on anassociation between a functional entity and a nucleic acid sequenceinformative about the structure of the functional entity. An advantageof such methods is that very large libraries can be constructed andprobed for a desired activity of the functional entities. Librarymembers having the desired activity can then be partitioned from librarymembers not having the desired activity, thus creating an enrichedlibrary with a higher fraction of members having the desired activity.This process is called selection. The structures of the library membersin the enriched library can then be identified by their cognate nucleicacid sequence, thus allowing identification even from minute amounts ofmaterial.

Some display technologies further allow the enriched library to beamplified without knowing the identity of its members; not merely thenucleic acid sequences but the functional entities too. Such displaytechnologies are called “amplifiable display technologies”. Thesetechnologies are especially advantageous when dealing with largelibraries, because iterative rounds of selection and amplification canbe performed allowing increased enrichment of desired activities.Another advantage of amplifiable display technologies is that rounds ofselection, amplification and diversification can be performed, thususing the same principle as in natural selection, to evolve moleculeswith desired function. This process is called molecular evolution.

Display technologies utilizing biological systems have been developed,the most notable of which is phage display (Smith, Science, 228, 1315-7,1985). However, such systems are limited to the display of naturaloccurring products such as proteins and peptides.

In vitro display technologies exploiting the flexibility of organicchemistry has been described. One example is described in U.S. Pat. No.5,723,598. The method uses a bifunctional molecule; one functionalitycapable of accepting a chemical group and one functionality capable ofaccepting a nucleic acid sequence. The method for synthesizing such alibrary is commonly known as “split and mix”, consisting of rounds ofmixing and splitting the bifunctional molecules into compartments. Acompartment specific pair of chemical group and nucleic acid sequence isadded. The nucleic acid sequences are thus encoding the chemical groups.All bifunctional molecules are then mixed and the process iterated tocreate a large combinatorial library. The library is then subjected toselection and the selected nucleic acid sequences amplified by PCR,which may be used for identification by conventional molecular biology;cloning and DNA sequencing.

Another example is described in WO04/039825A2, where a combinatoriallibrary is created, by rounds of proximity-guided addition of cognatepairs of chemical group and nucleic acid code to a bifunctionalmolecule; one functionality capable of accepting a chemical group andone functionality capable of accepting a nucleic acid sequence.Furthermore repertoires of so-called transfer units are used, where achemical group is attached to an oligonucleotide, containing a codingsegment and a segment capable of annealing to the bifunctional molecule.A repertoire of transfer units is annealed to the bifunctionalmolecules, which allows the code to be transferred enzymatically to thebifunctional molecule, as well as guiding the chemical group to reactwith the very same bifunctional molecule. This process can be reiteratedto create a large combinatorial library.

The libraries described above can be subjected to selections to form anenriched library. The enriched libraries members' synthetic history cansubsequently be deduced through the encoding nucleic acid. A limitationof these approaches is however that the enriched library cannot beamplified.

In vitro display technologies taking advantage of the flexibility oforganic chemistry and rounds of selections, amplification anddiversification has been described. These methods rely on the use oftemplates.

One example is described in WO00/23458, using a “split and mix’principle. A library of ssDNA templates is used, each containing achemical reaction site and several positions of codon segments. Thetemplates are compartmentalized by virtue of hybridizing to a repertoireof anti-codon sequences for a given codon position. Then, a compartmentspecific chemical reaction is performed modifying the reaction site onthe templates. The templates are then mixed and the process reiteratedby using other codon positions to form a combinatorial library.

Another example is described in WO02/074929A2, using a “single-pot”principle. A library of oligonucleotide templates is used, eachcontaining a chemical reaction site and several positions of codonsegments. Furthermore, using a repertoire of transfer units, the methodconsists of an oligonucleotide anti-codon sequence and a chemicalreactive group. The library of templates are hybridized with arepertoire of transfer units for a given codon position. This brings thechemical group on a hybridized transfer unit in proximity to thereaction site on the hybridized template, which consequently guides thechemical reaction of cognate pairs. This process is then reiteratedusing other codon positions to form a combinatorial library.

A limitation of the above proximity guiding of cognate pairs of code andchemical group is given by the linear structure of the templateoligonucleotide. As a consequence of the linearity the distance betweencodon and the chemical reaction site will differ from codon position tocodon position. For codon positions longer away from the reaction sitethe proximity guiding becomes compromised, as the local concentrationdrops to the power of three as a function of the distance. Thisdisadvantage becomes more pronounced for complex libraries, with morecodon positions and more complex codons.

This problem is sought solved in WO04/016767A2, where the transfer unitsbesides from an anti-codon segment also contain a constant segment,which is complementary to a constant sequence on the template close tothe reactive site. Thus, by hybridizing a transfer unit to a templateresults in that the template sequence between the codon position inquestion and the constant segment is bulging out, to form a so-calledomega structure. The concept is that the codon segment is responsiblefor the specificity and the constant segment responsible for theproximity. Also suggested in WO04/016767A2, is a so-calledT-architecture of the templates, where the reactive site on the templateis situated in the middle of the template, with the codon positionsspread out on each side. Consequently, the distance problem is so called“cut in half”.

WO 2004/056994A2 discloses a method similar to WO02/074929A2 orWO04/016767A2 with the difference that the template is cut into minorsequences, termed connecting polynucleotides in the application. Theconnecting polynucleotides connect transfer units to bring these intoreaction proximity. In certain embodiments the connectingpolynucleotides may comprise a reactive chemical group. To obtain anencoded molecule the method is dependent upon codon/anti-codonrecognition prior to reaction.

The template directed libraries described above are subsequentlysubjected to selections to form enriched libraries. The enrichedlibraries members' synthetic history can then be deduced through theencoding nucleic acid. The enriched libraries can also be amplified anddiversified by for example error prone PCR, thus allowing for molecularevolution.

A limitation in these approaches is that a large number of templateshave to be created, which is cumbersome, as the templates have to be ofconsiderable length to ensure proper codon/anti-codon hybridization. Inmethods using a plurality of minor sequences to make up the finaldirected synthesis the number of sequences to be synthesized is evenhigher than the actual library size.

The prior art methods using templates suffer from the disadvantage thatthe encoding is dependent upon hybridization of codon and anti-codonsequences. Sometimes hybridization between single strandedoligonucleotides will happen without perfect complementarities. In thecase of library construction the result is loss of the associationbetween the encoding and the synthetic history. Consequently, uponselection positive codes may be de-selected and negative codes may beselected. For more complex libraries this disadvantage becomes moresignificant as the complexity of the single stranded oligonucleotidesalso increases, both with respect to numbers, length and sequences. Thismakes the processes more difficult to control.

As described above in vitro display technologies allowing display of avariety of compound classes, selections, amplifications anddiversifications have been developed. However, there is still an ongoingneed for improvement, especially with respect to the quality in libraryconstruction and of diversification. The present invention offers amethod for producing an encoded molecule in which the method does notrequire the pre-synthesis of a large number of templates. Furthermore,the present method is not dependent upon codon/anti-codon recognitionfor an encoded molecule to be formed.

SUMMARY OF THE INVENTION

The present invention relates to in vitro display technology takingadvantage of the flexibility of organic chemistry, and permitting roundsof selection, amplification and diversification.

In particular, the present invention relates to a composition comprisinga nucleic acid and a chemical compound, said composition forming a starstructure defining 3 or more stems extending from a reaction center,wherein the stems are formed by a nucleic acid duplex and the chemicalcompound has been formed in the reaction center as the reaction productof 3 or more chemical groups.

The nucleic acid forms a super structure, in which different segmentshybridizes to each other so as to form a structure resembling a star.The star structure comprises a reaction center and a plurality of stems.In the reaction center, the chemical compound has been prepared as thereaction product of 3 or more chemical groups. The stems are nucleicacid duplexes, i.e. a stem comprises two hybridizing segmentscomplementing each other sufficiently for a duplex to be formed underconditions favoring the reaction of the chemical groups so as to formthe chemical compound.

At least one of the stems extent radially from the center. Suitably, the3 or more stems extent radially outwards from the reaction center. Theduplex nucleic acid of the stems is believed to bring the chemicalgroups together so as to obtain a reaction proximity. The proximityestablished between the chemical groups increases the localconcentration and enhances the chances for a reaction to proceed. Thepresence of 3 or more stems in the star structure creates a strong superstructure, which is stable even at conditions where a single duplex willseparate into two discrete single stranded nucleic acids. Thus, the starstructure enables versatile reaction conditions to be used in order topromote reaction between the chemical groups. Experiments reportedherein shows that the a three-stem star structure is stable enough fordirecting organic synthesis in medias containing in the excess of 35%acetonitril and tetrahydrofuran and in the excess of 40% DMF. In certainembodiments of the invention, the star structure encompasses 4, 5, 6, 7or more stems connected to a mutual reaction center. When the number ofstems is increased the stability of the star structure is alsoincreased.

The nucleic acid is segmented into various parts with certain functions.In certain aspect of the invention, the nucleic acid comprises one ormore codons identifying the one or more chemical groups, which haveparticipated in the formation of the formed chemical compound. Thepresence of a codon segment makes it possible not only to use thenucleic acid to promote reaction proximity but also to use the nucleicacid to code for one or more of the chemical groups which haveparticipated in the formation of the chemical compound. The presence ofone or more codons is especially useful for decoding purposes. When theformed chemical compound is present in a small amount or is present in amixture with other compounds, easy identification can be performedthrough molecular biological techniques.

A codon identifying a chemical group may be present anywhere in thenucleic acid forming the star structure, i.e. the codon may be presentat or in the vicinity of the reaction center, in the hybridizationsegments or in other parts of the star structure. In a certain aspect ofthe invention a codon is situated at the extremity of a stem. A codonplaced at the end of the stem pointing away form the center allows formore liberty in the design of the nucleic acid star structure as thecodon at the extremity does not necessarily need for take part in theformation of a duplex or in the formation of the environment for thereaction center.

The stems may be blunt ended, sticky ended or a loop may be present. Ablunt ended or sticky ended stem may be preferred when it is intended toligate the stem to another nucleic acid. In a certain embodiment, a loopis formed at the end of the stem. The loop forms a physical link betweenthe two strands thus forming a covalent linkage between the variousparts of the nucleic acid super structure. In a certain embodiment loopsare present at all extremes of the stems so as to form a circularnucleic acid. In another embodiment, a loop is present at all extremesof the stems except one, so as to form a contiguous nucleic acidsequence. Suitably, the contiguous sequence comprises a priming site toenzymatically extend the nucleic acid using a polymerase or anothernucleic acid active enzyme. In appropriate instances the priming site ispresent at the stem not having a loop. Suitably, the nucleic acidcomprises a priming site for a DNA polymerase, RNA polymerase or reversetranscriptase. Thus, the loops make it possible to prepare a doublestranded extension product displaying the formed chemical compound.

Importantly, the extension product comprises a generally linear duplex,i.e. a complementing strand has been formed by the extension reaction.The extension reaction destroys the reaction center so the chemicalcompound previously formed by reaction in the reaction center isdisplayed in the media. The display of the chemical compound enables theuse of various selection strategies on a library of extension products,as discussed later in this description.

Subsequent to the selection, the possibility of amplifying the nucleicacid is of particular relevance for identification purposes, because thechemical compound can be identified even in cases in which it occursonly in minute concentrations. The contiguous nucleic acid sequentsuitably comprises codons of all the reactants, which has participatedin the formation of the encoded chemical compound.

In a particularly preferred embodiment of the present invention a codonis situated in the non-base pairing part of the stem-loop structure. Thepresence in the loop of the codon allows for the use of any combinationof nucleotides in the design, as the specific sequence of a codon doesnot have material influence on the hybridization and reactioncapabilities.

In some aspects of the invention an enzymatic restriction site ispresent in the stem-loop structure. Depending on the specificendonuclease used, one or both strands of the stem-loop structure may bebroken. In a certain embodiment only one of the strands close to theloop is nicked, thereby forming a single stranded nucleic acid segment.The single stranded nucleic acid segment suitably contains the codon. Auseful enzyme for this purpose is N. Bbvc IA. In another embodiment bothstrands are broken and a sticky end, i.e. a single stranded nucleic acidoverhang, is formed. Suitably, the codon is present in the singlestranded overhang. In an aspect of the invention it is preferred to adda helper oligonucleotide complementary to at least to a part of thenucleic acid sequence of the loop. Under suitable conditions, the helperoligonucleotide hybridizes to the loop sequence and forms a substratefor a restriction enzyme.

The stems of the star structure may have any suitable length. Generally,a stem comprises two hybridisation segments having at least 80%complementarity and each hybridisation segment consists of 12 or morenucleotides. The complementarity is generally 90% or above, such as 95%or above. The hybridization segments may contain less than 12nucleotides for certain applications in which the stability of the starstructure may be dispensed with, such as 11, 10, 9, 8, 7, or 6nucleotides. However, in general a high stability is desired. A suitablestability under most conditions is generally obtained when eachhybridisation segment comprises 18 or more nucleotides. When conditionsare used which disfavor hybridization, i.e. temperatures well aboveambient, high salt concentrations, or presence of organic solvents,hybridization segments of 20 or more nucleotides are usually utilized.

After the reaction of the individual chemical groups, the formedchemical compound is preferably covalently attached to the nucleic acid.In certain applications it may be desired to use hybridization to attachthe formed chemical compound to the nucleic acid of the star structure;however a covalent attachment ensures that the chemical compound and thenucleic acid part remains together during a subsequent selection.

A chemical group to be reacted in the reaction center may be associatedwith the nucleic acid in any appropriate way. As an example, thechemical groups prior to reaction are covalently attached to the nucleicacid. Usually, one or more of the covalent attachments are cleavedsimultaneously with or subsequent to reaction. The covalent link may bedesigned to be cleavable or durable. Furthermore a cleavable linkage maybe designed to be cleaved immediately upon reaction or designed to becleaved in a step subsequent to a reaction.

Usually, the chemical compound is formed by reaction of the chemicalgroups attached to the nucleic acid and optionally one or more furtherreactants. The reactants may originate from any source, including be acompound added to the media as a free reactant not associated with anucleic acid. The further reactant(s) may be scaffolds, cross-linkingagents, activating agent, deprotecting agents etc.

The star structures according to the present invention are useful in thegeneration of libraries of different chemical compounds associated witha genetic code. Accordingly, the present invention also relates to alibrary of star structures. Each of the star structures may be presentin several copies in the media and the media generally comprises starstructures containing different chemical compounds. As an example, alibrary of the present invention may comprise at least 1000 differentchemical compounds, preferably 10⁶ different chemical compounds, andmore preferred 10⁹ different chemical compounds.

In another aspect, the present invention may be described as relating toe.g. a method for synthesizing large libraries associated with encodingnucleic acids through a “star-structure” formed by mutuallycomplementary oligonucleotides. This is obtained by hybridizingoligonucleotides containing two segments, where a segment towards the 3′end of one oligonucleotide hybridizes to a segment towards the 5′ in thenext and so forth. Finally, the segment towards the 3′ end of the lasthybridizes to a segment towards the 5′ end of the first oligonucleotide.Consequently, the mid section between the two hybridization segments oneach oligonucleotide is pointing towards the center of the formed ring,whereas the termini are pointing outwards, giving the star-structure.So, when three types of oligonucleotides are used three stems areformed, when four types of oligonucleotides are used four stems areformed etc. A chemical reactive group is associated to the mid sectionon each oligonucleotide, thus allowing proximity guided chemicalreactions to occur in the center. Furthermore, a codon is convenientlysituated external to the hybridized segment on each oligonucleotide,thus allowing encoding of the chemical groups participating in thecreation of the reaction product. The oligonucleotides with associatedchemical groups are called carrier modules herein. Consequently, acarrier module has a chemical group, two position specific segments anda codon. The formation of encoded combinatorial libraries is allowedwhen repertoires of carrier modules for each position are used.According to a certain embodiment, the assembled oligonucleotides aremade extendable or amplifiable when the termini in each stem, exceptone, are ligated via loop formations to form a continuousoligonucleotide with a 5′ and 3′ termini. Thus, consisting of one stemand a number of stem-loops, the star structure can be amplified by PCRor extended with a suitable enzyme. Consequently, the combinatorialdisplay library can be subjected to selection and the enriched librarymembers identified through their encoding oligonucleotide. Accordingly,an aspect of the invention relates to a method for creating one or morechemical structures comprising the steps of:

(i) providing N(N=3-100) carrier modules comprising:

-   -   (1) a first position carrier module having    -   i) a nucleic acid segment capable of hybridizing to a nucleic        acid segment of the N position carrier module, and    -   ii) a nucleic acid segment capable of hybridizing to a segment        of a second position carrier module,    -   (2) n position carrier module(s) (n=from 2 to N−1) having a        nucleic acid segment capable of hybridizing to said nucleic        acids segment of the n−1 carrier module, and a nucleic acid        segment capable of hybridizing to a segment of the n+1 carrier        module, and    -   (3) a N position carrier module having a nucleic acid segment        capable of hybridizing to said nucleic acid segment of said N−1        carrier module, and a nucleic acid segment capable of        hybridizing to a segment of said first carrier module, wherein    -   at least three of said carrier modules comprise an associated        chemical group (CG) situated in the mid section between the        hybridization segments or in the vicinity hereof and optionally        a codon segment situated external to one of the hybridization        segments;        (ii) contacting said carrier modules under conditions allowing        hybridization of said hybridization segments, thus bringing said        chemical groups into proximity, where the formed chemical        compound is associated with at least one of said carrier module.

N denotes the total number of carrier modules used in the formation ofthe chemical structure. Thus, when three carrier modules are used in theformation of the chemical structure, N is 3, when four carrier modulesare used in the formation of the chemical structure, then N is 4 etc. ndenotes the specific position of the carrier module.

Thus, when N is 3, a (1) first position carrier module is used having anucleic acid segment capable of hybridizing to a nucleic acids segmentof the third position carrier module, and a nucleic acid segment capableof hybridizing to a segment of a second position carrier module; (2)second (n=2) position carrier module is used having a nucleic acidsegment capable of hybridizing to said nucleic acids segment of thefirst carrier module, and a nucleic acid segment capable of hybridizingto a segment of third carrier module; and (3) a third position carriermodule is used having a nucleic acid segment capable of hybridizing tosaid nucleic acids segment of said second carrier module, and a nucleicacid segment capable of hybridizing to a segment of said first carriermodule.

When N is 4, a (1) first position carrier module is used having anucleic acid segment capable of hybridizing to a nucleic acids segmentof the fourth position carrier module, and a nucleic acid segmentcapable of hybridizing to a segment of a second position carrier module;(2) (n=2) second position carrier module is used having a nucleic acidsegment capable of hybridizing to said nucleic acids segment of thefirst carrier module, and a nucleic acid segment capable of hybridizingto a segment of third carrier module; and (n=3) third position carriermodule is used having a nucleic acid segment capable of hybridizing tosaid nucleic acid segment of the second carrier module and a nucleicacid segment capable of hybridizing to a segment of fourth carriermodule and (3) a fourth position carrier module is used having a nucleicacid segment capable of hybridizing to said nucleic acids segment ofsaid third carrier module, and a nucleic acid segment capable ofhybridizing to a segment of said first carrier module.

When N is 5, a (1) first position carrier module is used having anucleic acid segment capable of hybridizing to a nucleic acids segmentof the fifth position carrier module, and a nucleic acid segment capableof hybridizing to a segment of a second position carrier module; (2)(n=2) second position carrier module is used having a nucleic acidsegment capable of hybridizing to said nucleic acids segment of thefirst carrier module, and a nucleic acid segment capable of hybridizingto a segment of third carrier module; and (n=3) third position carriermodule is used having a nucleic acid segment capable of hybridizing tosaid nucleic acid segment of the second carrier module and a nucleicacid segment capable of hybridizing to a segment of fourth carriermodule and (n=4) fourth position carrier module is used having a nucleicacid segment capable of hybridizing to said nucleic acid segment of thethird carrier module and a nucleic acid segment capable of hybridizingto a segment of fifth carrier module; and (3) a fifth position carriermodule is used having a nucleic acid segment capable of hybridizing tosaid nucleic acids segment of said fourth carrier module, and a nucleicacid segment capable of hybridizing to a segment of said first carriermodule.

The above method may be followed by the step of providing conditionsallowing ligation of the termini of module n−1 to module n and moduleN−1 to module N, thereby forming a continuous nucleic acid molecule withstem-loop structures and a chemical compound associated. Thus, when N is3, a terminus of the fist module is allowed to ligate to a terminus ofthe second modules and a terminus of the second module is allowed toligate to the third module. When N is 4, (n=2) a terminus of the fistmodule is allowed to ligate to a terminus of the second module, (n=3) aterminus of the second module is allowed to ligate to the third module,and a terminus of a third module is allowed to ligate to the fourthmodule. When N is 5, (n=2) a terminus of the fist module is allowed toligate to a terminus of the second module, (n=3) a terminus of thesecond module is allowed to ligate to the third module, (n=4) a terminusof a third module is allowed to ligate to the fourth module, and aterminus of the fourth module is allowed to ligate to a terminus of thefifth module.

According to a further aspect of the present invention, the N positioncarrier modules may be ligated to the first carrier module, so as toform a circular nucleic acid.

A composition comprising a structure of nucleic acid and associatedchemical compounds made according to the method indicated above is novelsince carrier modules creates a novel “star structure” that aredifferent from the structures created in the prior art. See e.g. theprior art discussed in the Background section above.

In a preferred aspect of the invention a method is provided, whichensures a high proximity between the reaction chemical groups andamplification of the entire genetic code of the synthesis history of theformed chemical compound. Thus, in a preferred aspect, the presentmethod comprises contacting carrier modules under conditions allowinghybridization of hybridization segments, thus bringing reactive groupsinto reactive proximity; and

providing conditions allowing reaction of reactive groups, where theformed chemical compound is associated with at least one carrier module;and

conditions allowing ligation of the termini of module n−1 to module nand module N−1 to module N and thereby forming continuous nucleic acidmolecule with stem-loop structures and a chemical compound associated.

According to a preferred embodiment, N is 3, 4, 5, 6, 7. It is alsopreferred that each of the carrier modules comprise an associatedchemical group (CG) situated in the mid section between thehybridization segments or in the vicinity hereof and a codon segmentsituated external to one of the hybridization segments.

The contacting of the carrier modules may performed sequentially, i.e.the carrier modules may be contacted in any order between the individualcarrier modules or the contacting may be performed simultaneously, i.e.all the carrier modules, or at least a substantial amount of the carriermodules, are mixed together at hybridisation conditions so as to form asupermolecular complex. When sequential reaction of the chemical groupsis performed and only a fraction of the total amount of carrier modulesrequired for assembling the entire star structure is used, an auxiliaryoligonucleotide may be used to assemble a star structure, whereby thereaction center is formed. Thus, when a step in the formation of thechemical compound involves the assembling of two carrier modules byhybridization of the respective hybridization segments, an auxiliaryoligonucleotide having segments rovers complementary to thenon-hybridised hybridization segments may be added to form the starstructure.

After the chemical groups attached the carrier modules have been broughtinto close proximity in the reaction center, reaction is effected. Thechemical groups may be designed such that a reaction occurs immediatelywhen the groups come into reaction distance of each other or the groupsmay be designed such that an external component is necessary for thereaction to occur. The external component may be a reactant, a photon,electromagnetism or any other stimuli, which effects reaction. In acertain aspect of the present invention orthogonal chemistry is used,i.e. the chemical groups are designed such that the order of reaction isdirected.

The reaction center is defined by the stems surrounding said center. Ithas been suggested, that the distance between two reactants in thereaction center is less than 10 nm. Assuming the reaction center isspherical; the concentration of the reactants can be calculated to 1 mM.In a biological context a concentration of this size is regarded as highand a reaction can be assumed to proceed within a reasonable time.Furthermore, the concentration of free reactant in the media is verylow, when the carrier modules have been dosed in adjusted molar amounts,so the reaction in the reaction center is greatly favored overnon-directed reaction.

The mid section of the carrier module can contain any suitable chemicalgroups. To allow for enzymatic extension by e.g. a polymerase, the midsection of a carrier module suitably comprises a chemical bond or 1 to20 nucleotides. The nucleotides may be modified to obtain certainreaction conditions in the reaction center. As an example, thenucleotides of the mid section may be modified with lipophilic groups toprovide for a high mobility and reactivity of the associated chemicalgroups. The chemical group may be associated with the carrier moleculeat various positions. In one aspect, the chemical group is associatedwith a nucleobase of the mid section. In another aspect, the chemicalgroup is associated with a phosphodiester linkage of the midsection.

When the chemical group is attached to the backbone the point ofattachment is generally at the phosphor of the internucleoside linkage.When the nucleobase is used for attachment of the chemical group, theattachment point is usually at the 7^(th) position of the purines or7-deaza-purins or at the 5^(th) position of pyrimidines. The nucleotidemay be distanced from the reactive group of the chemical group by aspacer moiety. The spacer may be designed such that the conformationalspace sampled by the reactive group is optimized for a reaction with thereactive group of another chemical group in the reaction center. Ingeneral, the chemical group is associated to the midsection through oneor more covalent bonds.

The ligation may be effected prior to, simultaneously with or subsequentto reaction and the ligation maybe performed enzymatically or chemicallyat the choice of the experimenter. To reduce the amount of freenon-hybridising carrier modules in the media, it is generally desired toligate the carrier modules prior to reaction.

The formed chemical compound may be associated with the nucleic acidthrough a variety of chemical interactions. According to a preferredaspect the formed chemical compound is covalently associated with atleast one of said carrier molecules or the continuous nucleic acidmolecule. The relatively strong association between formed chemicalcompound and the nucleic acid, such as a covalent link, is useful duringthe screening of a library as it may be desired to us harsh conditions,which may disrupt weaker bonds, such as hydrogen bondings.

In a preferred aspect of the invention one or more carrier modules areprovided with a priming site for DNA polymerase, RNA polymerase orreverse transcriptase. The presence of a priming site assists in theamplification of the genetic code for the chemical group, which havereacted in the formation of the chemical compound. When ligation isabsent, i.e. the genetic code for each of the chemical groups remainsseparate entities kept together by hybridisation, it is preferred thateach carrier module contains a priming site. After a selection of alibrary has been performed, it is possible to gain informationconcerning which chemical groups that has participated in the formationof successful chemical compounds, i.e. chemical compounds with a desiredproperty. One way of obtaining this information is to quantify theamplification product, through well-know methods such as QPCR orstandard PCR combined with microarray. The information may be used inthe formation of a second-generation library with a reduced diversity,as it is only necessary to include carrier modules in the library, whichare successful. A reduced diversity library is also a focused librarybecause the abundance of chemical compounds with a desired property ishigher.

When two or more carrier modules are ligated together, it is possible toobtain information of the reactants, which together have participated inthe formation of chemical compounds with a desired property. Preferablyall, carrier modules are ligated together so as to form a linear nucleicacid or a circular nucleic acid. Thus, when two or more carrier modulesare ligated together, a single priming site is necessary to amplify thecontiguous nucleic acid comprising the two codons. According to apreferred embodiment, the method of the invention comprises a primingsite for a DNA polymerase, RNA polymerase or reverse transcriptase sitein at least the first carrier module and/or at least in the N carriermodule. When all the carrier modules are ligated together, i.e. thefirst carrier module is ligated to carrier module n (n=2 to N−1), whichis ligated to the carrier module N, a nucleic acid can be extended whena priming site is situated at one of the ends. To reduce the risk thatthe formed chemical compound remains hidden in the reaction center, anextension is preferably performed prior to the selection process. Theextension of the contiguous nucleotide effectively displays the formedchemical compound to the media and any target, which may be presenttherein.

Preferably, a priming site for hybridisation of a forward primer issituated at one end and a priming site for hybridisation of a reverseprimer is situated in the other end, so as to allow for amplificationaccording to the protocol of the polymerase chain reaction (PCR). PCRamplification is suitably performed after the selection has beenperformed to generate more copies of the genetic material of thestructures having the desired properties.

Accordingly, a second aspect of the invention relates to a compositioncomprising a structure of nucleic acid and associated chemical compoundor a library of more than one of such structures obtainable by themethod indicated above.

A library of chemical compounds associated with a nucleic acid codingfor the chemical groups, which have participated in the formation of thechemical compound, can be form by using a repertoire of carrier moduleson one or more positions.

The library as described herein may be used to screen for a compound ofinterest. It is generally desired to have a library as large as possibleto increase the possibility of finding a compound with desiredproperties. In a certain aspect of the invention the property of thecompound of interest is the ability to bind to a target. Generally, itis assumed that the possibility of finding a compound with high affinityand specificity towards a target is increasing with increasing librarysize. Thus, a library according to the present invention suitablycomprises more than 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³, or 10¹⁴ different chemical compounds associated with a nucleicacid encoding the synthetic history.

To obtain a library a repertoire of carrier modules may be used at anumber of positions. In an aspect of the invention, the repertoire on atleast one position comprises at least 10 different carrier modules. In acertain aspect, the repertoire on at least two positions comprises atleast 10 different carrier modules. To obtain a library of one milliondifferent chemical compounds in the same container, the multiplestructures of the invention can be formed with 100 carrier modules at 3different positions. In other words, synthesis of just 300 carriermodules enables the formation of a library of a million compounds.Similarly, a library of 100 million compounds can be formed with 100carrier modules at 4 different positions.

The invention also relates to a method for performing modulesubstitution. The method comprises the steps of:

a) providing a single stranded contiguous nucleic acid sequencecomprising N hybridisation segments and complementing hybridisationsegments as well as N−1 non-hybridising segments between thehybridisation segments and complementing hybridisation segments,b) hybridizing the nucleic acid under conditions favoring intramolecularhybridization, thereby forming a continuous nucleic acid, at leastcontaining N−1 stem-loops and one stem;c) introducing a break in said stem or loop thereby creating an overhangwhich at least contains a codon segment;d) providing a first group of carrier modules having at least: a nucleicacid segment capable of hybridizing to said stem, a nucleic acid segmentcapable of hybridizing to the stem of an adjacent stem-loop, optionallyan associated reactive group, and an anti-codon segment;d) providing conditions allowing hybridization of codon and anti-codonsegments; ande) providing conditions allowing enzymatical or chemical ligation ofsaid hybridized carrier module to the recessive termini of saidoverhang; and perform the steps of:

-   -   i) digest with a restriction enzyme the stem or loop of the        stem-loop adjacent to said codon sequence thereby making        overhangs which at least contain a next codon segment; and    -   ii) denaturate the nucleic acids; and    -   iii) hybridize under conditions favoring intramolecular        hybridization thereby forming N−1 stem-loops and one stem with        overhang at least containing said next codon segment; and    -   iv) optionally provide conditions allowing reaction of said        reactive groups, where the formed chemical compound is        associated with at least one of said carrier module; and    -   v) provide a next group of carrier modules having at least;    -   a nucleic acid segment capable of hybridizing to said stem, and        a nucleic acid segment capable of hybridizing to the stem of the        adjacent stem-loop, and optionally having a reactive group        associated, and having an anti-codon segment; and vi) provide        conditions allowing enzymatically or chemical ligation of        hybridized carrier module to the recessive termini of said        overhang; and repeat steps i) through vi) N−1 times; and        f) introducing a break in said stem-loop structure consisting        partial of said first group of carrier modules at least leaving        said anti-codon segment connected to said first carrier module;        and        g) denaturating the nucleic acids; and        h) hybridizing under conditions favoring intramolecular        hybridization thereby forming N−1 stem-loops and one stem; and        i) optionally, providing conditions allowing reaction of said        reactive groups, where the formed chemical compounds are        associated with at least one of said carrier module.

The contiguous nucleic acid sequence used in step a) may be providedfrom a number of sources. According to a first aspect, the nucleic acidis provided by the above method, however using dummy carrier modules notcarrying chemical groups. When these nucleic acids representing thecarrier modules are ligated together, a linear or circular nucleic acidis formed. According to a second aspect, the contiguous nucleic acidsequence of step a) is obtainable by performing an enzymatic extensionreaction to display the formed chemical compound. Thus, after theformation of the star structure, the single stranded extension productor a strand complementing the extension product may be used in step a).If desired, the extension product may be subjected to polynucleotideamplification, such as PCR, to amplify the number of copies of thenucleic acid.

In a certain aspect, the contiguous nucleic acid sequence in step a),b), or c) is obtained by immobilizing the sense strand of the PCRproduct on a solid support, isolating the solid support, allowing thesense stand to self-hybridize so as to form the star structure, and,optionally, breaking the stem attaching the self-hybridized starstructure with the solid support, thereby liberating the star structurefrom the solid support.

A suitable method of immobilizing the sense strand is to attach biotinto the primer producing the sense strand. The sense strand may then beattached to solid supports, such as beads, covered with streptavidin.Depending on the property of the solid support it may be isolated fromthe remainder of the media in a number of ways. Presently, it ispreferred to use magnetic beads, which easily can be isolated by amagnet. After the solid support has been washed a number of times, thesense strand is allowed to self-hybridize to form the star structureanew. In a preferred aspect, the re-folding is performed by instantcooling. The star structure may be maintained on the solid supportthroughout the module substitution process or the star structure may becleaved from the solid support. Suitably a cleavage of the stemattaching the solid support and the re-folded star structure isperformed with a restriction enzyme. The ability of the restrictionenzyme to perform the cleavage is actually a test confirming theintramolecular folding.

In certain aspects of the invention it may be suitable to cleave thestar structure in the loop. As most restriction enzymes recognize doublestranded nucleic acids only as substrates it is not immediately possibleto cleave in the loop using a restriction enzyme. Therefore, the presentinvention comprises the further step of adding a helper oligonucleotidecomplementary to a sequence of a loop, prior to a digesting step, tocreate a double stranded substrate for the restriction enzyme in theloop.

The invention also relates to a method for screening a library of morethan one chemical compound comprising:

probing the library for library members having a chemical compound ofdesired property; partitioning the library members having desiredproperty from library members not having desired property; and

thereby obtaining an enriched pool of library members having desiredproperty.

The enriched pool of library members having the desired property may beisolated and characterized if desired. However, one of the advantages ofthis method is that it is not necessary to isolated the enriched pool.In a preferred embodiment, the enriched pool is subjected to a nucleicacid amplification method to increase the genetic material indicative ofthe synthetic history of the chemical compounds having the desiredproperty. The pool of amplified nucleic acid representing the enrichedlibrary of compounds with a desired property may be decoded in order toidentify the reactants, which have been involved in the synthesis of thechemical compound with the desired property. However, if the enrichedpool is larger than it is feasible easy to decode the entire amount ofnucleic acid, the present invention offers the possibility ofreassembling the chemical compounds encoded by the enriched librarymembers or the nucleic acid representing such enriched library using theabove method of performing module substitution.

In one embodiment, the present invention provides a method foramplification of an enriched pool of library members having the desiredproperty. The method include that the PCR amplified oligonucleotides areallowed to hybridize under conditions favoring intramolecularhybridization, whereby the star-structure, consisting of a stem and anumber of stem-loops are recreated. The stem without a loop preferablycontains a recognition site for a restriction enzyme, which cuts outsideits recognition sequence and generates an overhang upon digest. Theredundancy of the sequence in the created overhang may conveniently beutilized to contain a codon. Restriction enzyme digestion of the stemthen generates codon specific overhangs for this first position. Therestriction enzyme digested star-structures are subsequently hybridizedwith a repertoire of carrier modules containing the two constantsegments for the first position and a cognate pair of the chemical groupand the anti-codon. Consequently, codon/anti-codon hybridizations allowappropriate pairs of carrier modules and star-structures to be ligatedby a DNA ligase. The neighboring stem-loop also contains a recognitionsite for another restriction enzyme capable of leaving a codon specificoverhang for this second position. Digestion with this secondrestriction enzyme thus eliminates the covalent linkage of the PCRamplified first module to the rest of the structure. The star-structuresare denatured and subsequently allowed to hybridize under conditionsfavoring intramolecular hybridization. The star-structures are therebyrecreated, but now with a new carrier module on position one (with anassociated chemical group) and the stem, without a loop is now locatedon position two. Rounds of this process may be performed to substituteall positions, to allow for proximity guided chemical reactions of theproper combinations of chemical groups. Consequently, rounds ofselection and amplifications can be performed until desired enrichmenthas been achieved.

The breaks in the stems may be introduced by a number of methods, suchas by restriction enzymes, e.g. RNase, Endonuclease III, endonucleaseVIII, APE1, Fpg, or by chemical cleavage or photo cleavage.

The contiguous nucleic acid sequence used in step a) of the modulesubstitution method described above can be provided by “breeding”. In acertain embodiment, a breeding method include the steps of:

digesting intermolecular hybridized nucleic acid structures derived froman enriched library with two consecutive restriction enzymes, whicheliminate the covalent linkages between the module in question and theremaining structure,

denaturing the digested structures,

allowing rehybridization of the nucleic acid fragments from the digestedstructures, thus allowing for exchange of a nucleic acid fractionspecifying the module in question to obtain breeding, and

ligation of the appropriate termini.

According to this method, carrier modules or nucleic acid partsrepresenting carrier modules can be shuffled. The shuffling allows for adiversification of the gene pool similar to breeding in a meioticbiological system. The method may be modified when nucleic acidfragments representing carrier modules, not present in the formation ofthe first generation library is added before allowing rehybridisation.Alternatively, the carrier module as such can be added before allowingrehybridisation. When new genetic material is added to the gene poolthis is similar to mutation in a biological system. The possibilities ofperforming breeding and mutation operations between generations oflibraries allow for an evolution strikingly similar to the naturalevolution process in the search for new drug candidates.

Thus, in a certain embodiment, the present invention provides a methodfor diversification of an enriched library, thus allowing molecularevolution. In the process described above for the amplification of adisplay library, a fraction of the library in each round for modulesubstitution, is digested with two consecutive restriction enzymes,which eliminate the covalent linkages between the module in question andthe remaining structure. The star-structures are denaturated andhybridized with a repertoire of carrier modules for the position inquestion. The position specific constant segments are thus guiding thehybridizations, equivalently to the creation of the primary library. Theappropriate termini are ligated and the formed product pooled with thecodon guided assembled fraction of the library, leading to adiversification. Consequently, rounds of selection, amplification anddiversification can be performed, thus allowing for molecular evolution.

In another embodiment, the present invention provides a method forbreeding of an enriched library, thus allowing molecular evolution. Inthe process described above for the amplification of a display library,a fraction of the library in each round for module substitution, isdigested with two consecutive restriction enzymes, which eliminate thecovalent linkages between the module in question and the remainingstructure. The star-structures are denatured and hybridized. Theposition specific constant segments are thus guiding the hybridizationsand allowing exchange of the module in question, i.e. breeding. Theappropriate termini are ligated and the formed product pooled with thecodon guided assembled fraction of the library, leading to adiversification. Consequently, rounds of selection, amplification,diversification and breeding can be performed, thus allowing formolecular evolution.

In another embodiment, the present invention provides a method forcreating combinatorial display libraries of polymers or small molecules.The chemical reactions may either be performed simultaneously orsequentially, by use of e.g. orthogonal chemistries, protective/maskinggroups, sequentially mixing of carrier modules, or carrier moduleswithout a CRG.

In one embodiment, the present invention provides a method for creatingcombinatorial display libraries of catalytic activity. In this aspectthe carrier modules are associated with reactive site functionalitiesand the star structure provides a framework for a three dimensionalarrangement of these functionalities.

The above-described aspects and embodiments show clear advantages overthe prior art. To name some, the various possible embodiments of thepresent invention show one or more of the following advantages: 1) aunique method for assembly of a combinatorial display library, 2) aunique structure for proximity guiding of chemical reactions, 3) thechemical reactions are highly independent of codon sequences becausethese are separated from the reactive site by constant segments, 4) highaccuracy in amplification of display library as only codons on arelevant position are capable of guiding fresh carrier modules as onlythese contain a termini to facilitate ligation, and 5) if accidentallyan incorrect codon/anti-codon guiding has occurred, the associationbetween encoding and display will still exist as the fresh carriermodules provides both the CRG and the code.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a discloses steps in the formation in the star structure, inwhich the chemical groups are simultaneously reacted.

FIG. 1 b shows steps in the formation of the star structure, in whichchemical groups are sequentially reacted.

FIG. 1 c discloses a method which uses convergent synthesis of thechemical compound.

FIG. 1 d shows a method in 5 steps for forming a library in which eachmembers displays the formed chemical compound efficiently.

FIG. 1 e discloses a method in which the loops are added after reactionof the chemical groups.

FIG. 2 a discloses a method for self-assembling of combinatorial libraryby repertoires of bi-specific oligonucleotides.

FIG. 3 shows 5 steps in an affinity selection.

FIG. 4 a discloses steps in the formation of a library.

FIG. 4 b discloses the principles of breeding and mutation of anenriched library.

FIG. 5 discloses steps in a method leading to the formation of anenriched library.

FIG. 6 shows the principle of molecular evolution.

FIG. 7 shows an embodiment of the invention involving an immobilizedsubstrate.

FIG. 8 discloses the experimental results of example 1.

FIG. 9 depicts the results of experiments reported in the example 2.

FIG. 10 shows the result of the experiments according to example 3.

FIG. 11 discloses the results of experiments reported in example 4.

FIG. 12 shows gels obtained in the experiments described in example 5.

FIG. 13 depicts a native PAGE gel from example 6

FIG. 14 shows a gel from the experiments reported in example 7

FIG. 15 discloses a gel of a re-folding experiment of example 8

FIG. 16 is a schematic diagram of the design of example 9.

FIG. 17 shows the gel resulting from the experiments reported in example9

FIG. 18 discloses the gel resulting from the experiments reported inexample 9

FIG. 19 shows various architectures of reaction design reported onexample 10.

FIG. 20 shows two gel resulting form experiments disclosed in example 12

FIG. 21 Discloses a gel from an experiment reported in example 13.

FIG. 22 shows a gel from an experiment reported in example 14.

FIG. 23 shows a gel from an experiment reported in example 15.

FIG. 24 discloses a gel from the experiment disclosed in example 16.

FIG. 25 shows the results of the experiment shown in example 17.

FIG. 26 discloses the outline of the experimental strategy used inexample 18

FIG. 27 shows a non-native PAGE gel of he individual steps in theprocess used in example 18.

FIG. 28 shows a non-native PAGE gel of the binding assay reported inexample 18.

FIG. 29 shows a PCR amplification gel reported in example 18.

FIG. 30 shows a picture of a gel evidencing the occurrence of areductive amination.

FIG. 31 discloses a picture of a gel evidencing the occurrence of ureaattachment

FIG. 32 shows gels of a study on the electromobility of the starstructure.

FIG. 33 shows a schematic representation of the translation process.

FIG. 34 shows the result of the experiments reported in example 22.

FIG. 35 shows the result of the experiments reported in example 22.

FIG. 36 shows the result of the experiments reported in example 22.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic Acid

Nucleic acid encoded chemical synthesis as described herein permits theproduction of combinatorial display libraries and the performance ofselection, amplification and evolution of a broad variety of chemicalcompounds such as small molecules and non-natural polymers. The nucleicacid serves multiple functions, for example, it brings chemicalreactants together, guides the three-dimensional arrangement of chemicalreactants, stores information regarding the chemical synthesis history,guides for proper matching of selected combinations of chemicalreactants and allows diversification and breeding of chemical compounds.

The method may be used to assemble one molecule, trillions of molecules,or even more at a time.

The method allows the isolation of ligands or drugs with propertiessuperior to those isolated by traditional rational design andcombinatorial drug discovery methods, as the chemical space can besystematically searched for ligands having desired properties.

Nucleic acid guided chemical synthesis has been shown to be awide-ranging phenomenon, not only limited to compounds of nucleic acidnature, but also applicable to guiding a broad range of chemicalreactions under a broad range of conditions (WO 2004/016767, WO2002/074929A2). This is of particular importance, as most molecules ofinterest do not resemble nucleic acid or nucleic acid analogs. Thechemical groups participating in the formation of the final chemicalcompound may be transferred in one step to a receiving chemical entityon a scaffold or a chemical group may be transferred in two steps, inwhich the first step includes a cross link between the chemical groupand the receiving entity and the second step include a cleavage of thechemical group from the carrier module to complete the transfer. Anexample of the former type of reaction of a reaction is a carrier modulehaving attached a 5-membered substituted N-hydroxysuccinimid (NHS) ringserving as an activator, i.e. a labile bond is formed between the oxygenatom connected to the NHS ring and the chemical group to be transferred.The chemical groups can be transferred to a recipient nucleophilicgroup, typically an amine group, which may be present on a scaffold. Theremainder of the fragment is converted into a leaving group of thereaction. When the chemical group is connected to the activator througha carbonyl group and the recipient group is an amine, the bond formed onthe scaffold will an amide bond.

An example of a two-step reaction is the so-called allylglycin reaction.In a first step a chemical group comprising a carboxylic acid or aderivative there of is reacted with a nucleophilic group, such as anamine. The chemical group is attached to an allylglycin group, which ina second step may be cleaved with iodine to release the chemical group.The two-step reaction method is disclosed in more detail in WO2004/039825, the content thereof being incorporated herein by reference.Another example of a two-step reaction strategy is shown in more detailin example 10.

Of pivotal importance for nucleic acid guided synthesis of combinatorialdisplay libraries is the proximity guiding of reactants, which ensuresreaction efficiency and proper association of encoding and display.Proximity of reactants is obtained by associating together thereactants, by some sort of linker. Proximity can also be described as alocal concentration, which is dependent on the length and flexibility ofthe linker. If free flexibility of the linker is assumed, the localconcentration can be calculated by using the volume of a sphere with thelinker length as radius. The formula to calculate the volume of a sphereis; v=4/3*pi*r³. Consequently, the proximity or local concentrationdrops in the 3^(rd) power as a function of the linker length. Forexample a linker length around 10 nm, will be equivalent to aconcentration around 1 millimolar, whereas a 100 nm linker will beequivalent to a concentration around 1 micromolar. Efficient organicchemistries are typically performed in the millimolar to molarconcentration range. Consequently, to ensure efficiency in the chemicalreactions the linker length should not be significantly longer than 10nm.

Preferably, the reactive groups are brought into reactive proximity ofless than 100 nm, more preferably less than 50 nm, even more preferablyless than 25 nm, even more preferably less than 10 nm and mostpreferably less than 5 nm.

In the prior art for single-pot synthesis of DNA encoded displaylibraries allowing amplification, single stranded DNA templates withcodons spread out over the length of template are used (WO 2004/016767,WO 2002/074929A2). The templates are responsible for recruiting transferunits having proper anti-codon sequences from a repertoire of transferunits and thereby bringing together chemical groups on the template andthe transfer unit. Consequently, the single stranded template acts alsoas a linker between the chemical group on the template and the chemicalgroup on the transfer unit hybridized to the template. Hence, the linkerlength and thereby the local concentration of reactants will depend onwhich codon position is employed. An unfolded (extended) oligonucleotidehaving for example 20 nucleosides will have a length around 10 nm, (thesix-bond backbone spacing is around 0.63 nm) and an oligonucleotidehaving 200 nucleosides will have a length around 100 nm. Consequently,unfolded oligonucleotides considerably longer than 20 nucleosides (10nm, equivalent to a concentration around 1 millimolar) will in generalnot be suitable to create proximity guiding of chemical reactions.

In one embodiment the present invention circumvents the lengthenedstructure of nucleic acid in use to bring reactants into reactionproximity. This is achieved by choosing appropriate sequences ofoligonucleotides capable of folding into stable three dimensionalstructures and thereby allowing proximity guiding by sequence positionsseparated by many nucleosides. As shown in FIG. 1 a this is achieved byusing bi-specific oligonucleotides (mutually complementary), which canhybridize into a “star-structure”. The bi-specific oligonucleotidescontain two segments: a segment towards the 3′ end of oneoligonucleotide hybridizes to a segment towards the 5′ in the next andso forth. Finally, the segment towards the 3′ end of the last hybridizesto a segment towards the 5′ end of the first oligonucleotide.Consequently, the mid section between the two segments on eacholigonucleotide is pointing towards the center. This mid section can bea bond or a segment. In contrast, the termini are pointing outwards,thus giving the star-structure. So, when three types of oligonucleotidesare used three stems are formed, when four types of oligonucleotides areused four stems are formed etc. A chemical reactive group (CRG) isconveniently associated to or in the vicinity of the mid section on eacholigonucleotide. The chemical reactive groups are thus brought intoreaction proximity, as the diameter of the DNA double helix is around 2nm, thus allowing proximity guided chemical reactions to occur in or inthe vicinity of the center.

The chemical reaction is performed such that the formed product isassociated to at least one oligonucleotide. Furthermore, a codon isconveniently situated external to one or both of the hybridized segmentson each oligonucleotide, thus allowing encoding of the chemical groups.The oligonucleotides with associated chemical group, two positionspecific hybridisation segments and a codon are called carrier modules.

To make the created combination of oligonucleotides amplifiable by e.gPCR, the termini in each stem, except one, are ligated via loopformations to form a continuous oligonucleotide with a 5′ and 3′termini. In one aspect, the structure consists of one stem and a numberof stem-loops, which can be amplified by having PCR priming sites at thetermini (FIGS. 1 d and 1 e). Alternatively, all termini are ligatedforming a closed ring, which may be amplified by primer extension by aDNA polymerase without strand displacement activity.

A method using stepwise reaction of the chemical groups are shown inFIG. 1 b. Initially, two carrier modules are contacted underhybridisation conditions. Carrier module A comprises a hybridisationsegment a, which anneals to hybridisation segment a′ of carrier moduleB. Following the annealing step, a chemical reaction between chemicalgroup C_(A) on carrier module A and C_(B) on carrier module B isallowed. In a third step, carrier module C is added under hybridisationconditions. Carrier module C comprises a hybridisation segment b′, whichcomplements the hybridisation segment d of carrier module B. Thereaction proximity of the product C_(A)-C_(B) to the chemical groupC_(C) enables the chemical reaction to proceed so as to produce theproduct C_(A)-C_(B)-C_(C). A fourth carrier module D is added underhybridisation conditions. Carrier module D is allowed to hybridize tothe growing star structure, so as to bring reactant C_(D) into closeproximity of reaction product of the preceding reaction, whereby areaction is promoted to produce the final chemical compoundC_(A)-C_(B)-C_(C)-C_(D).

FIG. 1 c discloses a convergent synthesis method, in which the initialreaction steps follow two separate pathways. In a first series ofreactions carrier modules A and B are contacted separately underhybridisation conditions in a first container. Hybridisation segment aof carrier module A and hybridisation segment a′ of carrier module Bwill anneal to each other and a reaction between the chemical groupsC_(A) and C_(B) is effected. In a second container carrier modules C andD are mixed under hybridisation conditions so as to form a hybridisationproduct in which hybridisation segment c of carrier module C is annealedto hybridisation segment c′ of carrier module D. Subsequently, reactionoccurs between chemical group C_(C) and chemical groups C_(D) to produceintermediate product C_(C)-C_(D). The intermediate products are allowedunder hybridisation conditions to anneal to each other, whereby thestart structure is formed. Subsequently, or simultaneously with theformation of the star structure, reaction between the two intermediatereaction products C_(A)-C_(B) and C_(C)-C_(D) is allowed to produce thefinal chemical compound C_(A)-C_(B)-C_(C)-C_(D). When performingstepwise or convergent synthesis it may be useful prior to reaction ofthe chemical groups to provide an auxiliary oligonucleotide to form thereaction center.

In one embodiment, the present invention relates to the formation of theloop in the stem-loop structures as provided by carrier modules as shownin FIG. 1 d. Alternatively, the loops are formed by ligation ofstem-loops provided by other oligonucleotides as shown in FIG. 1 e orany combination hereof of the embodiments shown in FIGS. 1 d and 1 e.The terminal carrier modules may contain PCR priming sites or thepriming sites may be provided by ligating other oligonucleotides.

The embodiment disclosed in FIG. 1 d is shown in five steps. In thefirst step, four carrier modules, each carrying a reactive groups R andbi-specific oligonucleotides are contacted under hybridisationconditions. The carrier modules are in equilibrium with the starstructure. In the second step the hybridisation complex is ligated atthe termini of the carrier modules so as to form a continuous nucleicacid. The proximity of the chemical groups at the center of the starstructure promotes the reaction and in step 3 a product is formed, whichis attached with a linking entity to the nucleic acid coding for thechemical groups which have participated in the formation of the chemicalcompound. In step 4 a priming site for a polymerase is ligated to thestar structure to enable extension of the nucleic acid. The last stepshows the extension product in which a double stranded DNA has beenformed using the nucleic acid of the star structure as a template.

In FIG. 1 e, a variant of the embodiment of FIG. 1 d is shown, as thestem-loop is added separately and ligated to the star structure. In afirst step, four carrier modules are mixed. Due to the existence ofhybridisation segments, the star structure is formed under hybridisationconditions. Subsequent to formation of the hybridisation complex,reaction is effected to form the chemical compound. After formation ofthe compound by reaction of the four chemical groups, 3 stem-loops and apriming site for a polymerase is added. The stem-loops and the primingsite comprise an overhang which complements an overhang of the starstructure. When a ligase is added, the stem-loops and the priming siteare ligated to the start structure, so as to form a continuous nucleicacid.

In certain embodiments the present invention relates to ligation ofcarrier modules, for example using enzymes such as T4 DNA ligase, TaqDNA ligase, T4 RNA ligase or E. coli DNA ligase or by chemical ligation(Shabarova et al., Nucleic Acids Res, 19, 4247-51, 1991).

Carrier Modules—Oligonucleotide Portion

Oligonucleotides are used to guide the chemical reactions in the presentinvention. The oligonucleotides in this context are called carriermodules, which contain at least two position specific hybridizationoligonucleotide segments, optionally an oligonucleotide codon segment,and a reactive chemical group.

In one embodiment the present invention relates to carrier modules,where the oligonucleotide portion consists of DNA, RNA or analogs hereofand in any combinations hereof. The oligonucleotide portion is capable,at least after modification, of being an appropriate template instandard protocols for nucleic acid replication and/or amplifications.

The carrier modules may be synthesized using methodologies known in theart. For example the oligonucleotide may be prepared by any method knownin the art for synthesizing oligonucleotides, e.g. solid phase synthesisusing an automated synthesizer. Oligonucleotides following synthesis maybe associated when desired (for example, covalently or non-covalentlycoupled) with a CRG of interest using standard coupling chemistriesknown in the art.

In one embodiment the present invention relates to carrier modules,where the association of the CRG to the oligonucleotide is to the midsection between the hybridization segments or in the vicinity hereof.The mid section may be a phosphordiester linkage, derivatives thereof ora nucleic acid segment. In vicinity of the mid section relates tolocations on the duplex nucleic acid stem, preferentially to locationsclose to the mid section. Preferably, the vicinity of the mid sectionrelates to less than 20 nucleotides, more preferably less than 10nucleotides, even more preferably less than 5 nucleotides and mostpreferably less than 2 nucleotides.

In one embodiment the present invention relates to carrier modules,where an association of a CRG to an oligonucleotide occurs via linkersor spacers, which are long and flexible enough to allow the reactants tocome into reaction proximity. The linkers preferentially have a lengthand composition to permit reactions between reactants paired byoligonucleotides, but yet minimizing reactions with unpaired entities.Moreover, the association between the oligonucleotide and the CRG may bethrough a covalent bond. In certain embodiments, the covalent bond maybe more than one.

The linkage can be cleavable by for example light, oxidation,hydrolysis, exposure to acid, exposure to base, or reduction. A varietyof linkages useful in the practice of the invention is described in theprior art (Fruchtel and Jung, Angew Chem Int Ed Engl, 35, 17, 1996). Thelinker assists contact of the reactants and in certain embodiments,depending on the desired reaction, positions DNA as a leaving group,where the linker is cleaved as a natural consequence of the reaction. Incertain embodiments depending on the desired circumstances reaction ofone reactive group is followed by cleavage of the linker attached to asecond reactive group to yield products without leaving behindadditional atoms capable of providing chemical functionality.

In one embodiment the present invention relates to carrier modules,where the association of the CRG to the oligonucleotide occurs throughthe backbone of the nucleic acid

In one embodiment the present invention relates to carrier modules,where the association of the CRG to the oligonucleotide is through thebase. In a preferred embodiment the CRG is associated to thenon-Watson-Crick hydrogen bonding parts.

In one embodiment the present invention relates to carrier modules,where the association of the CRG to the oligonucleotide allows readthrough by a DNA polymerase, at least after its removal.

In one embodiment the present invention relates to carrier modules,where the association of the CRG to the oligonucleotide is non-covalent.For example if biotin is attached to the oligonucleotide andstreptavidin is attached to the CRG, hence an interaction between biotinand streptavidin associates the oligonucleotide and the CRG with eachother non-covalently.

Carrier Modules—Chemistry

A broad range of compounds and/or libraries of compounds can be preparedusing the methods described herein. In certain embodiments, compoundsthat are not, or do not resemble, nucleic acids or analogs thereof, aresynthesized according to the method of the invention. In certain otherembodiments, compounds that are not or do not resemble, proteins oranalogs thereof, are synthesized according to the method of theinvention.

In one embodiment the present invention relates to sequential chemicalreactions of proximity guided reactants. For example, by use oforthogonal chemistries or the use of orthogonal protective/maskinggroups, or by sequential assembly and reaction of carrier molecules.

The assembly of carrier modules without ring formation, i.e. formationof a contiguous nucleic acid, may by itself bring appropriately locatedCRGs into proximity, as the diameter of a double helix is around 2 nmthus allowing positioning of several consecutive CRGs within reactionproximity. The reaction conditions, linkers, reactants and reaction siteare chosen to avoid non-oligonucleotide guided reactions and accelerateoligonucleotide guided reactions. Sequential or simultaneouslycontacting of carrier molecules can be employed depending on theparticular compound to be synthesized. In a certain embodiment ofspecial interest, the multi-step synthesis of chemical compounds iscontemplated in which three or more carrier molecules are contactedsequentially to facilitate multi-step synthesis of complex chemicalcompounds.

In one embodiment the present invention relates to annealing of carriermodules, which allows the use of carrier modules at concentrations lowerthan concentrations used in many traditional organic synthesis. Thuscarrier modules may be used in submillimolar concentrations. Preferably,the carrier module concentrations may be used in submillimolarconcentrations of less than 100 micromolar, more preferably less than 10micromolar, even more preferably less than 1 micromolar, even morepreferably less than 100 nanomolar and most preferably less than 10nanomolar

In one embodiment the present invention relates to CRG forming smallmolecules or polymers. Known chemical reactions for synthesizingpolymers or small molecules can be used in the practice of the presentinvention. The chosen reactions preferably are compatible with nucleicacids, such as DNA and RNA or analogs thereof. Reactions useful include,for example, substitution reactions, carbon-carbon bond formingreactions, elimination reactions, and addition reactions.

The CRG or reactants include a variety of reagents and can be anychemical group or reactive moiety (e.g. electrophiles, nucleophiles)known in the chemical art.

In synthesizing small molecules using the method of the presentinvention a carrier module may have a scaffold associated upon which thesmall molecule is to be assembled. The scaffold can be any chemicalcompound with two or more sites for functionalization. The sites may beprotected by methods and protecting groups known in the art. Theprotecting groups may be orthogonal to each other so that they can beremoved individually. The reactants to modify a scaffold may be, forexample electrophiles (e.g. acetyl, amides, acid chlorides, esters,imines), nucleophiles (e.g. amines, hydroxyl groups, thiols) or sidechains.

In certain embodiments, polymers, specifically unnatural polymers, aresynthesized according to the method of the present invention. Theunnatural polymers that can be synthesized using the inventive methodand system include any unnatural polymers. For example unnaturalpolymers include, but are not limited to, peptide nucleic acid (PNA)polymers, polycarbamates, polyureas, polyesters, polyacrylate (e.g.polyethylene, polypropylene), polycarbonates, polypeptides withunnatural stereochemistry, polypeptides with unnatural amino acids, andcombination thereof. In certain embodiments, the polymers comprise atleast 3, 4, 5, 6, 7, 8, 9, 10, 25 monomer units or more. In certainembodiments the monomer units may comprise di-mers, tri-mers ortetra-mers or oligomers. The polymers synthesized using the inventivesystem may be used, for example, as catalysts, pharmaceuticals ordiagnostic affinity ligands. In preparing certain unnatural polymers,the monomer units are attached to the carrier module. The monomer unitsmay be, for example, carbamates, D-amino acids, unnatural aminoacids,PNAs, ureas, hydroxy acids, esters, carbonates, acrylates, or ethers. Incertain embodiments, the monomer units have two reactive groups used tolink the monomer unit into the growing polymer chain. Preferably, thetwo reactive groups are not the same so that the monomer unit may beincorporated into the polymers in a directional fashion, for example, atone end may be an electrophile and at the other end a nucleophile.Reactive groups may include, but are not limited to, esters, amides,carboxylic acids, activated carbonyl groups, acid chlorides, amines,hydroxyl groups, and thiols. In certain embodiments, the CRGs are maskedor protected. (Green et al (1999) Protective Groups in Organic Synthesis3^(rd) Edition, Wiley) so that polymerization may not occur until adesired time when the CRGs are deprotected. Once the monomers arebrought together via carrier module assembly, initiation of thepolymerization results in a cascade of polymerization and deprotectionsteps wherein the polymerization results in deprotection of a reactivegroup to be used in the subsequent polymerization step. The monomerunits to be polymerized can include two or more monomers.

The monomer units may contain any chemical groups known in the art.Reactive chemical groups especially those that would interfere withpolymerization, hybridization, etc., are preferably masked using knownprotecting groups ((Green et al (1999) Protective Groups in OrganicSynthesis 3^(rd) Edition, Wiley). In general, the protective groups usedto mask these reactive groups are orthogonal to those used in protectingthe groups used in the polymerization steps.

In one embodiment the present invention relates to carrier modules,where the reactive site is associated with the same carrier module forall chemical reactions. For example a small molecule scaffold isassociated with one carrier module and the remaining carrier modulesprovide entities modifying the scaffold.

In one embodiment the present invention relates to carrier modules,where the reactive site will shift positions during the chemicalreactions

In one embodiment the present invention relates to the association ofthe formed chemical compound to the oligonucleotide while maintainingread through by a DNA polymerase for example at least after its removal.

Preparation of Combinatorial Library

An important practical difference between traditional and nucleic acidguided library synthesis is the scale of each manipulation. Due to theamounts of material needed for screening and compound identification,traditional combinatorial syntheses typically proceed onnanomol-micromol scale per library member. In contrast, nucleic acidguided library synthesis can take place on the femptomol-picomol scalebecause only minute quantities (e.g. about 10⁻²⁰ mol) of each nucleicacid-linked synthetic molecule are needed for selection and PCRamplification. The vast difference in scale, combined with thesingle-solution format in nucleic acid guided library synthesissimplifies significantly the preparation of materials required.

In one embodiment, the present invention relates to the formation of acombinatorial display library. Libraries can be produced by use ofrepertoires of carrier modules on some or on all positions (FIG. 2 a).In a first step a repertoire of carrier modules for each position isprovided. When the carrier modules are mixed under hybridisationconditions, they will assemble into the star structure, directed by thesequence of the hybridisation segments. After assembling of the carriermodules ligation and reaction is effected in any order. In an aspect ofthe invention, the ligation is performed before the reaction to increasethe stability of the star structure. Subsequent to the proximity guidedreaction, a polymerase priming site is ligated to the star structure andan extension reaction is preformed to display the formed chemicalcompound to the exterior environment.

As would be appreciated by one skilled in this art, libraries of smallmolecules and polymers can be synthesized using the principles disclosedherein. Consequently, the combinatorial display library can be subjectedto selection and the enriched library's members identified through theirencoding oligonucleotide.

Depending upon the circumstances repertoires of carrier modules for twoor more positions are initially combined and subjected to a nucleic acidguided chemical reactions between the attached CRGs. Depending upon thecircumstances the library can be formed by multiple chemical reactions,wherein each intermediate product is purified before the subsequentround of reactions. Preferably less than 20 chemical reactions steps arerequired to create a library. In other embodiments, less than 10chemical reaction steps are needed, and more preferably between 3 and 9steps are needed to create a the library

Selection

Selection and/or screening for reaction products with desired activities(such as catalytic activity, binding affinity, binding specificity, or aparticular effect in an activity assay) might be performed according toany standard protocol. For example, affinity selections (see FIG. 3) maybe performed according to the principles in library-based methods suchas phage display (Smith, Science, 22S, 1315-7, 1985), ribosome display(Hanes et al., Proc Natl Acad Sci USA, 95, 14130-5, 1998), mRNA display(Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-302, 1997) orDNA encoded chemical libraries (WO 2004/016767, WO 2002/074929A2).Selection for catalytic activities may for example be performed byaffinity selection on transition state analog affinity columns (Baca etal., Proc Natl Acad Sci USA, 94, 10063-8, 1997) or by function basedselection schemes (Pedersen et al., Proc Natl Acad Sci USA, 95, 10523-8,1998). Since minute quantities of DNA (˜100 molecules) can be amplifiedby PCR, these selections can thus be conducted on a scale of thismagnitude allowing a truly broad search for desired activities, botheconomical and efficient.

The display library can be selected or partitioned for binding to atarget molecule. In this context, selection or partitioning means anyprocess whereby a library member bound to a target molecule is separatedfrom library members not bound to target molecules. Selection can beaccomplished by various methods known in the art. In most applications,binding to a target molecule preferable is selective, such that thebinding to the target is favored over other binding events. Ultimately,a binding molecule identified using the present invention may be usefulas a therapeutic reagent and/or diagnostic agent.

The selection strategy can be carried out to allow selection againstalmost any target. Importantly, the selection strategy does not requireany detailed structural information about the target molecule or aboutthe members of the display library. The entire process is driven by thebinding affinities and specificities involved in library members bindingto a given target molecule.

Selected library members can easily be identified through their encodingnucleic acid, using standard molecular biology. The present inventionbroadly permits identifying binding molecules for any known targetmolecule. In addition, novel unknown targets can be discovered byisolating binding molecules of selected library members and use thesefor identification and validation of a target molecule.

Selection of binding molecules from a display library can be performedin any format to identify binding library members. Binding selectiontypically involve immobilizing the desired target molecule, adding thedisplay library, allowing binding, and remove non-binders/weak-bindersby washing. The enriched library remaining bound to the target may beeluted with, for example acid, chaotropic salts, heat, competitiveelution with known ligand, high salt, base, proteolytic release oftarget, enzymatic release of nucleic acids. In some embodiments theeluted library members are subjected to more rounds of binding andelution, using the same or more stringent conditions or using adifferent binding format, which will increase the enrichment. In otherembodiments the binding library members are not eluted from the target.To select for library members that bind to a protein expressed on a cellsurface, such as an ion channel or a transmembrane receptor, the cellsthemselves can be used as selection agents. A selection procedure canalso involve selection for binding to cell surface receptors that areinternalized so that the receptor together with the binding moleculepasses into the cytoplasm, nucleus, or other cellular compartment, suchas the Golgi or lysosomes. Isolation of the compartment in questionleads to partitioning of library members being internalized fromnon-internalized library members (Hart et al., J Biol Chem, 269,12468-74, 1994). A selection procedure may also involve in vivoselection. For example by in vivo organ targeting, where a library isinjected into an animal and the organ of interest is subsequentlyisolated and thereby obtain an enriched pool of library members targetedto that organ (Pasqualini and Ruoslahti, Nature, 380, 364-6, 1996). Theenriched library's nucleic acid portion may be amplified by, for examplePCR, leading to many orders of amplification, allowing identification bye.g cloning and DNA sequencing.

According to the specific embodiment for affinity selection shown inFIG. 3, a library of reaction products resulting from the embodimentshown in FIG. 2 a, is contacted with a target under binding conditions.If one or more of the formed chemical compounds have affinity towardsthe target a binding will result. In a subsequent step, binding librarymembers or a nucleic acid derived therefrom are partitioned. The nucleicacid attached to the formed chemical compound is subsequently amplifiedby e.g. PCR to produce multiple copies of the nucleic acid, which codesfor the synthesis history of the compound displaying the desiredaffinity. The amplified nucleic acid can be sequenced by a number ofwell-known techniques to decode which chemical groups that haveparticipated in the formation of the successful compound. Alternatively,the amplified nucleic acid can be used for the formation of a nextgeneration library.

Other Selections

Selections for other properties, such as catalytic or other functionalactivities, can also be performed. Generally, the selection should bedesigned such that library members with a desired activity can beseparated from other library members. For example, selection for librarymembers with capacity for catalyzing bond cleavage can be performed byhaving biotin attached to each library member by the bond in question.Partitioning using streptavidin can then separate library members havingthe catalytic activity from others. Another example is selection forlibrary members with bond formation capabilities. This can be performed,by attaching a substrate to each library member and subsequently addinga substrate to which biotin is attached. A reaction between the twosubstrates forming a bond will attach biotin to catalytic librarymembers. Partitioning using streptavidin can then separate librarymembers having the catalytic activity from others. Selection for otherproperties, such as dimerization and/or polymerization may also beperformed. In this case library members can be partitioned by size ofthe formed complex, using for example, HPLC, acrylamid or agarose gelsor size exclusion columns.

Nucleic Acid Amplification

Amplification of the nucleic acid portion of enriched library membersmay be performed by standard protocols for nucleic acid amplification.These methods include, for example, polymerase chain reaction (PCR)(Saiki et al., Science, 230, 1350-4, 1985), nucleic acid sequence-basedamplification (NASBA) (Compton, Nature, 350, 91-2, 1991), stranddisplacement amplification (Nycz et al., Anal Biochem, 259, 226-34,1998), self-sustained sequence replication (Mueller et al., HistochemCell Biol, 108, 431-7, 1997), primer extension, and plasmidamplification (see for example Sambrook, J., Fritsch, E F, and Maniatis,T. (1989) in: Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory.

Assembly of Display Library by Module Substitution

In one embodiment, the present invention relates tore-assembly/amplification of an enriched library member or a secondgeneration library of enriched library members to re-create the display.In the case of an enriched library an enriched display library isformed, thus, allowing rounds of selection and amplification andre-assembly. For example, as shown in FIG. 4 a, the above described PCRamplified oligonucleotides of enriched library members are allowed tohybridize under conditions favoring intramolecular hybridization,whereby the star-structure, consisting of a stem and a number ofstem-loops is recreated. The stem without a loop may contain arecognition site for a restriction enzyme, which generate an overhangwith ambiguous base(s). The ambiguous base(s) of the sequence in thecreated overhang is conveniently utilized to contain a codon.Restriction enzyme digestion of the stem thus generates codon specificoverhangs for this first position. The restriction enzyme digestedstar-structures are then hybridized with a repertoire of carrier modulescontaining the two constant segments for the first position and acognate pair of CRG and anti-codon. Consequently, codon/anti-codonhybridization allows for appropriate pairs of carrier modules andstar-structures to be ligated. The neighboring stem-loop may alsocontain a recognition site for another restriction enzyme capable ofleaving a codon specific overhang for this second position. Digestionwith this second restriction enzyme thus eliminates the covalent linkageof the PCR amplified first module to the rest of the structure. Thestar-structure is denaturated, and subsequently allowed to hybridizeunder conditions favoring intramolecular hybridization. Thestar-structures are thereby recreated, but now with a new carrier moduleon position one (with a CRG), and the stem, without a loop, is nowlocated on position two. Rounds of this process are performed tosubstitute all positions, to allow for proximity guided chemicalreactions of the proper combinations of CRGs; the display library isthereby amplified and re-assembled. Finally PCR priming sites may beligated to the star-structure. Consequently, rounds of selection andamplifications and re-assembly can be performed until desired enrichmenthas been achieved (see FIG. 5).

In one embodiment, the present invention relates to the formation ofcodon specific overhangs created by restriction enzymes. Suitablerestriction enzymes are capable of forming overhangs with more than onespecific sequence. Such enzymes include i) restriction enzymes withambiguous bases in their recognition sequence, ii) restriction enzymescutting outside their recognition sequence and iii) restriction enzymesperforming nicks (nicking endonucleases). Examples of such restrictionenzymes; AlwNI, ApaBI, Asul, BbvI, BbvII, BccI, Bce831, Bcefl, BciVI,BglI, BinI, BseMII, BseRI, BsgI, BsiYI, BsmAI, BspMI, BsrDI, BstEII,BstXI, BtgZI, DdeI, DraII, DraIII, DrdI, Earn1105I, EciI, Eco31I,Eco57I, Eco57MI, EcoNI, EspI, Esp3I, Fnu4HI, Fold, GsuI, Hinfl,Hpy178III, Hpy188I, Ksp632I, MaeIII, MboII, MmeI, MwoI, PflMI, PfoI,PleI SapI, SauI, ScrFI, SecI, SfaNI, SfiI, Sth132I, Tsp4CI, TspDTI,TspGWI, TspRI, Tth111I, Tth111II, XcmI, N.AlwI, N.BstNBI, N.BbvCIA andN.BbvCIB.

The encoding capacity (the number of different codons possible) of anoverhang is given by the number of ambiguous bases in the overhangcreated by the restriction enzyme. Hence, for every N(N=A, T, G or C)four different residues can be chosen, for every H (H=A, C or T), V(V=A, C or G), B (B=C, G or T) and D (D=A, G or T) three differentresidues can be chosen and for every R(R=A or G), K (K=G or T), Y (Y=Cor T), S(S=C or G) and M (M=A or C) and W (W=A or T) two differentresidues can be chosen. Consequently, the encoding capacity iscalculated by multiplying the number of different residues on eachposition with each other. For example Sfi I;

(SEQ ID NO: 1) 5′- . . . GGCCNNNN/NGGCC . . . -3′ (SEQ ID NO: 1)3′- . . . CCGGN/NNNNCCGG . . . -5′is creating the overhang 5′-NNN-3′, thus consisting of three Ns, thushaving an encoding capacity of 64 (=4×4×4).

Another example, Ava II;

5′- . . . G/GWCC . . . -3′ 3′- . . . CCWG/G . . . -5′creating the overhang 5′-GWC-3′, thus having an encoding capacity of 2.

Another example is Bbs I;

(SEQ ID NO: 2) 5′- . . . GAAGACNN/NNNN . . . -3′ (SEQ ID NO: 3)3′- . . . CTTCTGNNNNNN/ . . . -5′creating an overhang consisting of four Ns, thus having an encodingcapacity of 256 (=4×4×4×4).

A special group of restriction enzymes are those restriction enzymescutting only one strand (nicking endonuclease). These enzymes may inprinciple have indefinite encoding capacity; for example, when i) therecognition sequence is located in the stem of a stem-loop structure, orii) used to create a terminal overhang, or iii) in the combination withanother restriction enzyme. This is because the length of the createdoverhang can in principle be of indefinite length.

For example, N. BbvC IA located in the stem of a stem-loop structure;

a digest results in;

5′- . . . CC -3′ (SEQ ID NO: 6) 3′- . . . GGAGTCGNNNNNNCGACT-5′in this example six Ns are present in the formed overhang, thus givingan encoding capacity of 1024 (=4×4×4×4×4×4). However, it's apparent thatthe number of Ns can be chosen arbitrarily thus giving an indefiniteencoding capacity. A small fraction of the total number of possiblesequences in this example can't be used. Namely those sequences forminga recognition sequence of the restriction enzyme in use.

Furthermore a nicking endonuclease can create a terminal overhang ofarbitrary length, for example

N. BbvC IA

(SEQ ID NO: 7) 5′- . . . CC/TCAGCNNNNNNNN- 3′ (SEQ ID NO: 8)3′- . . . GGAGTCGNNNNNNNN- 5′a digest results in;

5′- . . . CC -3′ (SEQ ID NO: 8) 3′- . . . GGAGTCGNNNNNNNN- 5′ and(SEQ ID NO: 9) 5′-TCAGCNNNNNNNN- 3′

In this example eight Ns are present in the formed overhang, thus givingan encoding capacity of 65536 (=4 in the 8^(th) power). However, it'sapparent that the number of Ns can be chosen arbitrarily thus giving anindefinite encoding capacity. A small fraction of the total number ofpossible sequences in this example can't be used. Namely those sequencesforming a recognition sequence of the restriction enzyme in use.

Furthermore a nicking endonuclease in combination with a restrictionenzyme can be used to create overhangs of arbitrary length without anyrequirements for a stem-loop structure. For example

N. BbvC IA combined with Eco RI;

(SEQ ID NO: 10) 5′- . . . CC/TCAGCNNNNNNNNG/AATTC . . . -3′(SEQ ID NO: 11) 3′- . . . GGAGTCGNNNNNNNNCTTAA/G . . . -5′a digest results in;

5′- . . . CC -3′ (SEQ ID NO: 12) 3′- . . . GGAGTCGNNNNNNNNCTTAA- 5′ and;(SEQ ID NO: 13) 5′- TCAGCNNNNNNNNG -3′ and 5′- AATTC -3′ 3′- G -5′in this example eight Ns are present in the formed overhang, thus givingan encoding capacity of 65536 (=4 in the 8^(th) power). However, it'sapparent that the number of Ns can be chosen arbitrarily thus giving anindefinite encoding capacity. A small fraction of the total number ofpossible sequences in this example can't be used. Namely those sequencesforming a recognition sequence of the restriction enzyme in use.

Although the length of the codon segments may vary, the codon segmentsmay range from 1 to 50 nucleotides, from 1 to 40, from 1 to 30, from 1to 15, from 1 to 10 Codon segments, however, preferentially are 2, 3, 4,5, 6, 7, 8, 9 or 10 nucleotides long.

Although the length of the stem forming segments may vary, the stemsegments may preferentially range from 5 to 50 nucleotides, from 5 to40, from 5 to 30, from 5 to 15, from 5 to 10. Stem segments, howeverpreferentially are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long

The length of the mid section between the stem forming segments mayvary. The mid section may preferentially range from a singlephosphodiester bond (or analogue bond) to a stretch of 20 nucleotides.However, the mid section is preferentially a single phosphodiesterbondor 1, 2, 3, 4, 5, or 6, nucleotides long.

In one embodiment the present invention relates to a method forre-assembly/-amplification of a display library, where thestar-structures following restriction enzyme digest further are treatedwith a phosphatase, which removes the 5′ phosphate and thus preventligation of two star-structures. Several suitable phosphatases are knownin the art, for example antarctic phosphatase and calf intestinalalkaline phosphatase.

In one embodiment the present invention relates to a method forre-assembly/-amplification of a display library, where the carriermodules contain a 5′ phosphate to facilitate ligation to thestar-structure.

In one embodiment the present invention relates to a method forre-assembly/amplification of a display library, where the carriermodules are phosphorylated after ligation to a star-structure. Thisprevents ligation between free carrier modules.

In certain embodiments the present invention relates to a method forre-assembly/amplification of a display library, where the PCR amplifiedstar-structure's 5′ terminus may be created by other means thanrestriction enzymes for example; RNase, Endonuclease III, endonucleaseVIII, APE1, Fpg, chemical cleavage or photo cleavage. A PCR productconsists of a primer in the 5′ end and the remaining sequence formed bya DNA polymerase. The primer may contain residues not found in thesegment formed by the DNA polymerase, such as dUTP or RNA. Such residuesmay be specifically recognized and cleaved by appropriate means, whichwill create a defined terminus (Smith et al., PCR Methods Appl, 2,328-32, 1993).

In one embodiment the present invention relates to a method forre-assembly/amplification of a display library. The PCR amplifiedenriched library termini may be modified before the formation of astar-structure, by any of the above mention methods.

Diversification

In one embodiment, the present invention contemplates a method fordiversification of a displayed compound or library of displayedcompounds, thus allowing molecular evolution. This can be achieved in anumber of ways without going beyond the scope of the present invention.For example (see FIG. 4 b), a fraction of the molecules in a round formodule substitution is digested with two consecutive restrictionenzymes, which eliminate the covalent linkages between the module inquestion and the remaining structure. The star-structures aredenaturated and hybridized with a repertoire of carrier modules for theposition in question. The position specific constant segments are thusguiding the hybridizations, in the same way as the primary library wascreated. The appropriate termini are ligated and the formed productpooled with the codon guided assembled fraction, leading todiversification. This may be done in one, some or all rounds of modulesubstitution. In another example, a fraction of the molecules in a roundfor module substitution is subjected to removal of codon specificoverhangs for the position in question, e.g. by an exonuclease.Subsequently a repertoire of carrier modules for the position inquestion is hybridized and ligated. The then formed non-codon guidedproducts are pooled with the codon guided assembled fraction, leading todiversification. This may be done in one, some or all rounds of modulesubstitution.

The diversification may also be performed by shuffling/recombination(breeding) of modules between library members before the modulesubstitution process. For example, the enriched library members' nucleicacid portion is amplified and digested with a restriction enzyme cuttingin constant segment(s), thus creating two or more fragments. Thefragments can be ligated with fragments originating from other librarymembers to form a full length product, whereby shuffling/recombinationshave occurred. Another example of methods for shuffling/recombinationsis by using the star-structures (see FIG. 4 b). The star-structures aredigested by two consecutive restriction enzymes, denaturated and allowedto hybridize leading to exchange of the module in question.Consequently, rounds of selection, amplification and diversification canbe performed, thus allowing for molecular evolution (see FIG. 6).

Selection for Catalytic Activity

The principle described can also be applied to select for catalyticactivity. In this case the carrier modules include reactive sitefunctionalities and the star structure provides a framework for a threedimensional arrangement of these functionalities, thus mimicking proteinenzymes.

Selection schemes for various catalytic activities are contemplated. Forexample i) selection for binding to a transition state analog, ii)selection for bond formation by associating one substrate to the starstructures while the other substrate is immobilized to e.g. beads.(Consequently library members associated to the beads are capable ofbond formation), or iii) selection for bond cleavage by having thesubstrate associated to both the star structure and a bead.(Consequently library members not associated to the bead are capable ofbond cleavage.) (see FIG. 7).

Codon Specific Compartmentalization

The star-structure allows any codon position to become terminal andsingle stranded by use of for example a suitable restriction enzyme,thus allowing highly specific compartmentalization by hybridization andoptionally ligation for a particular codon position. Various methods forcompartmentalization are known in the art, for example, microarrays ofanti-codon sequences (Lockhart et al., Nat Biotechnol, 14, 1675-80,1996), columns of anti-codon sequences (Halpin and Harbury, PLoS Biol,2, E173, 2004) or using beads, where the individual beads contain ananti-codon sequence and a fluorescence tag, which subsequently allowsfor sorting by e.g. fluorescence activated cell sorted (Iannone et al.,Cytometry, 39, 131-40, 2000).

Such compartmentalization may be useful in the practice of the presentinvention, for example; i) during library synthesis for post chemicalreaction modifications, ii) analysis of single clones, iii) analysis ofprogression in selections, or iv) analysis of diversity. Consequently,compartmentalization in situation ii)-iv) may be a rapid and economicalalternative to DNA sequencing for deconvolution of a single sequence ora library of sequences.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present invention also consistessentially of, or consist of, the recited components and that theprocesses of the present invention also consist essentially of, orconsist of, the recited processing steps. Further, it should beunderstood that the order of steps or order for performing certainactions are immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The method and compositions of the present invention represent new waysto generate molecules with desired properties. This approach combinesextremely sensitive and powerful molecular biology, with the flexibilityof organic chemistry. The ability to prepare, amplify, and evolveunnatural polymers and small molecules by molecular evolution may leadto new classes of catalysts, novel ligands, or drugs with superiorproperties to those isolated with slower traditional discovery methods.

The present invention also provides kits and composition for the use inthe inventive methods.

DEFINITIONS

The terms, “nucleic acid” or “oligonucleotide” as used herein refer to apolymer of nucleotides. The polymer may include, without limitation,natural nucleosides (i.e. adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine; anddeoxycytidine), nucleoside analogs, (eg., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-urouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,biologically modified bases (e.g. methylated bases), intercalated bases,modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose and hexose), or modified phosphate groups (e.g.,phosphorothioates and 5′, -N-phosphoramidite linkages). Nucleic acidsand oligonucleotides may also include other polymers of bases having amodified backbone, such as a locked nucleic acid (LNA), a peptidenucleic acid (PNA), a threose nucleic acid (TNA) and any other polymerscapable of serving as a template for an amplification reaction, using anamplification technique, for example, a polymerase chain reaction or aligase chain reaction.

The term “segment” as used herein, refers to a continuous section of anoligonucleotide sequence.

The terms, “codon” and “anti-codon” as used herein, refer to anoligonucleotide sequence that code for a certain chemical groupassociated with the said codon or anti-codon. A series of codons codesfor the combination of specific chemical reactants, which haveparticipated in the formation of the encoded molecule.

The term “Stem-loop” structure as used herein, refers to any secondarystructure involving at least a nucleotide portion within which a strandof a nucleic acid sequence, via intramolecular hydrogen bonds, withanother portion of the same nucleic acid molecule in order to constitutea “self-paired” region termed “stem” of mostly double-stranded natureand an unpaired “loop” region located at one end of the said stem. Whenthe length of the loop is zero, it produces the special case ofstem-loop called “hair pin” or palindrome.

The term “small molecule” as used herein refers to an organic compoundeither synthesized in the laboratory or found in nature having amolecular weight less than 10,000 grams per mole, optionally less than5,000 grams per mole, and optionally less than, 2,000 grams per mole,such as less than 1000 grams per mole. Preferred small molecules aresuitable for oral administration.

The terms, “small molecule scaffold” or “molecular scaffold” as usedherein, refer to a chemical compound having at least one site orchemical moiety suitable for functionalization. The small moleculescaffold or molecular scaffold may have two, three, four, five or moresites or chemical moieties suitable for functionalization. Thesefunctionalization sites may be protected or masked as would beappreciated by one skilled in this art. The sites may also be found onan underlying ring structure or backbone.

The terms “chemical reactive group” or “chemical groups” or “reactiveunit” as used herein, refer to any chemical moiety capable of modifying,adding to, or taking away from another chemical moiety. Including, forexample, but not limited to, a building block, monomer, monomer unit,molecular scaffold, or other reactant useful in proximity mediatedchemical synthesis. In some instances the chemical group is not anucleotide or a derivative thereof. In another aspect at least one ofthe chemical groups which have participated in the synthesis of theformed chemical compound is not a naturally occurring amino acid.

The term, “associated with” as used herein describes the interactionbetween or among two or more groups, moieties, compounds, monomers, etc.When two or more entities are “associated with” one another as describedherein, they are linked by a direct or indirect covalent or non-covalentinteraction. Preferably, the association is covalent. The covalentassociation may be, for example, but without limitation, through anamide, ester, carbon-carbon, disulfide, carbamate, ether, thioether,urea, amine, or carbonate linkage. The covalent association may alsoinclude a linker moiety, for example, a photocleavable linker. Desirablenon-covalent interactions include hydrogen bonding, van der Waalsinteractions, dipole-dipole interactions, pi stacking interactions,hydrophobic interactions, magnetic interactions, electrostaticinteractions, etc. Also, two or more entities or agents may be“associated” with one another by being present together in the samecomposition.

The term, “carrier module” as used herein, refers to a chemical groupassociated to an oligonucleotide, and a segment towards the 3′ end whichcan hybridize to a segments towards the 5′ in a second oligonucleotideand a segment towards the 5′ end which can hybridize to a segmenttowards the 3′ end of a third oligonucleotide. The carrier moduleoptionally contains a codon or anti-codon segment. The term“hybridization segment” as used herein refers to said oligonucleotidesegment.

The term “star-structure” as used herein, refers to any secondarystructure involving at least three stems of mostly double strandednature. 0, 1, 2, 3, 5, 6, 7, 8, 9 or more nucleotide residues mayseparate the stems. In the special case where zero nucleotide residuesare separating four stems the junction is called a Holliday junction. Astar structure may consist of one nucleic acid molecule, or it mayconsist of a plurality of nucleic acid molecules.

The term “reaction proximity” as used herein, refers to a distancebetween reactants by which the reaction of said reactants can occur in acontrolled, efficient and timely manner.

The term “proximity guided chemical reaction” as used herein, refers tochemical reactions between reactants, which are brought into reactionproximity by hybridization of nucleic acid to which the reactants areassociated.

EXAMPLES Example 1 The Formation of Trimeric and Tetrameric DNA StarStructures by Mutual Complementary Bi-Specific Oligonucleotides isDemonstrated

DNA oligonucleotides (prepared by DNA Technology Århus, Denmark) weremixed as indicated in the table shown in FIG. 8 in 2 uM concentrationseach in 1× Ligase Buffer (New England Biolabs), 50 mM NaCl. The mixtureswere incubated at 80 degrees C. for 2 minutes and slowly cooled to roomtemperature in a water bath. The products were analyzed by PAGE native(7.5% polyacrylamide), followed by staining with ethidium bromide, usingstandard protocols (Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989)in “Molecular Cloning: a Laboratory Manual”, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring, Harbor, N.Y.).

Oligo6 and oligo7 (corresponding to Vip006 and vip007, respectively),oligo6 and oligo8 (corresponding to vip006 and vip008) and oligo7 andoligo8 (corresponding to vip007 and vip008, respectively) each have amutual complementary segment, thus capable of forming an annealed dimer.Accordingly, a band corresponding to dimers were observed in lane 1-3.Vip006, vip007 and vip008 each have a mutual complementary segment totwo neighboring oligoes, thus capable of forming a closed annealedtrimeric structure (see FIG. 8). Accordingly, a band corresponding to atrimer was observed in lane 4. Oligo6, oligo7, and oligo9 (correspondingto Vip006, vip007 and vip009, respectively) each have a mutualcomplementary segment to a neighboring oligo, thus capable of forming anannealed trimeric structure. However, the structure is open becausevip008 and vip006 do not have complementary segments (see FIG. 8).Accordingly, a band corresponding to a trimeric was observed in lane 5.The open trimer is expected to have a slightly lower mobility in the gelthan the more compact closed trimer form. The mobility difference is infact observed when comparing lane 4 with 5.

An equivalent observation of a slow migrating trimeric band was obtainedby using oligo6, oligo7, and oligo 10 (corresponding to vip006, vip007and vip010, respectively), where vip006 and vip010 do not annealdirectly to each other (compare lane 4 and 6). To assess the efficiencyof formation of the closed trimeric form oligo6, oligo7, and oligo8(corresponding to vip006, vip007, and vip008, respectively) wereannealed in the presence of two-fold excess of oligo9 or oligo10(corresponding to vip009 or vip010, respectively). Interestingly, themajor band in both lanes 7 and 8 correspond to the closed trimeric fastmigrating species consisting of vip006, vip007 and vip008. Thesuccessful formation of a closed tetramer was accomplished by annealingoligo6, oligo7, oligo9 and oligo10 (corresponding to vip006, vip007,vip009 and vip010, respectively) and observed as one major band in lane9. Note that the intended valency in all chases were obtained with highefficiency; observed as a single major band.

Example 2 Conversion of Trimer DNA Star Structure into a SingleContiguous Strand of DNA by T4 DNA Ligase

The successful creation of a tri-stemmed DNA star structure consistingof a single uninterrupted strand of DNA was demonstrated in thisexample. Mutual complementary bi-specific oligonucleotides wereannealed, and subsequently ligated to form a continuous strand of DNA.

DNA oligoes (prepared by DNA Technology Århus, Denmark) were mixed asindicated in FIG. 9 in 2 uM concentrations each in 1× Ligase Buffer (NewEngland Biolabs), 50 mM NaCl. The mixtures were incubated at 80 degreesC. for 2 minutes and slowly cooled to room temperature in a water bath.

The 5′ termini of the oligonucleotides were phosphorylated by T4 DNApolynucleotide kinase. A mixture consisting of 1.67 uM star structure,1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl and 0.2 u/ul T4DNA polynucleotide kinase (New England Biolabs, cat# M0201), wasprepared and incubated for 30 minutes at 37° C.

A phosphodiester bond between juxtaposed ends of annealedoligonucleotides was formed by T4 DNA ligase (New England Biolabs, cat#M0202). ⅓× Volume of ligase mix, 1×DNA ligase Buffer (New EnglandBiolabs), 50 mM NaCl, and 100 u/ul T4 DNA ligase (New England Biolabs,cat# M0202), were added to the above described kinase treated mixtureand incubated for 2 hours at room temperature. The products wereanalyzed by non-native PAGE (7.5% polyacrylamide, SM urea), followed bystaining with ethidium bromide, using standard protocols (Sambrook, J.,Fritsch, E. F. and Maniatis, T. (1989) in “Molecular Cloning: aLaboratory Manual”, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring, Harbor, N.Y.).

Vip076 (25 nt) and vip017 (42 nt) each have a mutual complementarysegment which upon annealing places the 3′ end of vip076 adjacent to the5′ end of vip017, thus making a substrate for T4 DNA ligase.Accordingly, a prominent band corresponding to vip076-vip017 (67 nt) wasobserved in lane 1 (non-native PAGE). Similarly, the formation of aprominent band corresponding to vip017-vip078 (100 nt) was observed inlane 3. In contrast, vip076 and vip078 (58 nt) each have a mutualcomplementary segment but do not form annealed adjacent ends and are notexpected to be ligated. Accordingly, only bands corresponding tomonomers of vip076 and vip078 were observed in lane 2. Note the bandcorresponding to vip076 is fainter, which is expected as vip076 issmaller and the oligonucleotides are in equimolar concentrations.Moreover, vip078 (58 nt) migrates slower than vip076-vip017 (67 nt) inthe gel, which is not unexpected as vip076-vip017 contains sequences forcreation of a stem-loop structure giving a more compact fold, thushigher mobility in the gel.

Vip076, vip017 and vip078 each have a mutual complementary segment,which upon annealing places the 3′ end of vip076 adjacent to the 5′ endof vip017, and the 3′ end of vip017 adjacent to the 5′ end of vip078,thus making two substrates for T4 DNA ligase. Accordingly, a prominentband corresponding to vip076-vip017-vip078 was observed in lane 4.

Consequently, creation of a trimeric DNA star structure consisting ofone contiguous strand of DNA was hereby demonstrated.

Example 3 Amplification of Tri-Stemmed DNA Star Structure

The successful amplification of trimeric DNA star structure consistingof one contiguous strand of DNA was demonstrated in this example. Mutualcomplementary bi-specific oligonucleotides were annealed, ligated andsubsequently used as a template in a primer extension reaction.

DNA oligoes (prepared by DNA Technology Århus, Denmark) were mixed asindicated below in 2 uM concentrations each in 1× Ligase Buffer (NewEngland Biolabs), 50 mM NaCl. The mixtures were incubated at 80 degreesC. for 2 minutes and slowly cooled to room temperature in a water bath.

The 5′ termini of the oligonucleotides were phosphorylated by T4 DNApolynucleotide kinase. A mixture consisting of 1.67 uM star structure,1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl and 0.2 u/ul T4DNA polynucleotide kinase (New England Biolabs, cat# M0201), wasprepared and incubated for 30 minutes at 37 C

A phosphodiester bond between juxtaposed ends of annealedoligonucleotides was formed by T4 DNA ligase (New England Biolabs, cat#M0202). ⅓× Volume of ligase mix, 1×DNA ligase Buffer (New EnglandBiolabs), 50 mM NaCl, and 100 u/ul T4 DNA ligase (New England Biolabs,cat# M0202), were added to the kinase treated mixture and incubatedovernight at room temperature.

A primer extension reaction was performed by adding 3 volume extensionmix, 1.33× Vent Buffer (New England Biolabs), 1.33 uM vip038, 2.67 mMdNTP and with or without 1.33 u/ul Vent(exo-) DNA polymerase (NewEngland Biolabs, cat# M0257) to 1 volume ligation reaction. The solutionwas incubated for 1 minutes at 92 C, 1 minute at 50 C and 10 minutes at74 C and put on ice.

The reactions were analyzed by native PAGE (7.5% polyacrylamide)followed by staining with ethidium bromide, using standard protocols(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) in “MolecularCloning: a Laboratory Manual”, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring, Harbor, N.Y.).

The results of the experiments are shown in FIG. 10. Vip038 is reversecomplementary to the 20 most 5′ terminal bases in vip078 and cantherefore prime an extension reaction using vip078 or vip078 fusions astemplates. Accordingly, a single prominent band corresponding to doublestranded vip073 was obtained in the extension reaction containingvip038, vip076 and vip078 (lane 2). Note that this band is not presentin lane 6, which is equivalent but without the DNA polymerase included.In contrast, lane 6 contains two bands, which presumably consists ofannealed vip076/vip078/vip038 and annealed vip078/vip038.

The specificity of the reaction was demonstrated by vip038, vip076 andvip017, where no visible difference between with or without the DNApolymerase was observed, compare lane 1 and 5.

A successful primer extension was also observed using the ligationreaction vip017-vip078 illustrated by the prominent band in lane 3. Notea fainter band corresponding to double stranded vip078 also is observedillustration that not all vip078 was ligated to vip017. In thecorresponding lane 7 without DNA polymerase a fainter band with almostthe same mobility as double stranded vip017-vip078 is observed. The bandpresumably consists of annealed vip038/vip017-vip078.

A successful primer extension was also observed usingvip076-vip017-vip078 ligation reaction as template. Two bands with lowermobility in the gel than double stranded vip017-vip078 were observed.The lower band corresponds to annealed vip038/vip076-vip017-vip078 asseen when comparing to the equivalent lane 8 without DNA polymerase.However, the upper band in lane 4 is unique and therefore consisting ofdouble stranded vip076-vip017-vip078. Consequently, this exampledemonstrates that DNA structures can be converted into double strandedDNA and therefore amplifiable.

Example 4 Chemical Reactivity in the Center of Star Structures

The chemical reactivity in the center of star structures wasaccomplished by using the following tri-functional cross-linker (TSAT,Tris-succinimidyl aminotriacetate, Pierce cat. No 33063):

The DNA oligoes: vip016/vip017 or vip016/vip017/vip018 (prepared by DNATechnology Århus, Denmark), all having an internal amino modified dT(GlenResearch, cat. No.: 10-1038-xx) were mixed in 150 mM NaCl, 100 mMsodium phosphate, pH 7.2 giving 20 uM total oligo concentration. Themixtures were incubated at 80 degrees C. for 2 minutes and slowly cooledto room temperature in a water bath. TSAT was dissolved in DMF. A 10fold serial dilution in DMF was prepared. 1 volume DMF or TSAT dilutionwas mixed with 9 volume buffer giving a final buffer concentration of150 mM NaCl, 100 mM sodium phosphate, pH 7.2. 1 volume buffered DMF orbuffered TSAT dilution was mixed with 1 volume annealed DNA oligomixture giving final oligo:TSAT ratios; 1:0, 1:10, 1:100, 1:1000, 1;10000, and allowed to incubate for 2 hours at room temperature. Thereactions were analyzed by PAGE; both native (7.5% polyacrylamide), aswell as by non-native PAGE (7.5% polyacrylamide, 8M urea), followed bystaining with ethidium bromide, using standard protocols (Sambrook, J.,Fritsch, E. F. and Maniatis, T. (1989) in “Molecular Cloning: aLaboratory Manual”, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring, Harbor, N.Y.).

Vip016 and vip017 each have a mutual complementary segment, thus capableof forming an annealed dimeric structure. Vip016, vip017 and vip018 eachhave a mutual complementary segment to the neighboring oligo, thuscapable of forming a closed annealed trimeric structure (see FIG. 12).

As expected in lanes 1-5, native gel, the major band corresponds to adimeric structure, whereas the major band in lanes 6-10 in the nativegel corresponds to a trimeric structure. In a non-native gel theannealing is disrupted. As expected a band in lanes 1 and 6 in thenon-native gel corresponding to a monomer was observed. However, whenTSAT was included higher order structures were observed (lanes 2-5,7-10, non-native gel) indicating that TSAT did cross-link the oligoes.Interestingly, when only vip016 and vip017 were present the highestorder cross-linked structure was dimeric (non-native gel, lanes 2-5),whereas when vip016, vip017, vip018 were present an additional trimericstructure was observed (non-native gel, lanes 7-10), thus indicatingthat cross-linking is dependent on annealing of the DNAoligonucleotides. Noteworthy is also that as expected a bell-shaped doseresponse was observed; at low TSAT concentration the reaction will beslow leading to poor yields, as the TSAT concentration increases thereaction will proceed faster leading to more cross-linked product,however at higher

TSAT concentrations the cross-linking will be competed by TSAT moleculesreacting with only one DNA oligo leading to lower cross-linked product.Hence, the highest yield of cross-linked product was observed using 1000TSAT equivalents (lanes 4 and 9, non-native gel). Moreover, theadvantageous in a closed annealed structure for cross-linking wasillustrated by the much higher overall yields obtained in lanes 7-10when compared to their counterparts in lanes 2-5 with the same TSATconcentration.

Example 5 Chemical Reactions Directed by DNA Star Structure

The oligonucleotide, vip017, was functionalized by cross-linking anamino acid (L-Leu or Gly Fluka, #61820 and #50052) through thealfa-amine to the primary amine on an internal modified dT in vip017, bythe homobifuntional linker BSOCOES((Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), Pierce cat#21600) bytreatment of the oligonucleotide (5 nmol) in a 200 mM pH 7.4 sodiumphosphate solution (200 uL) with 0.1 volumes of a 100mM BSOCOES solutionin DMF for 10 min at 25° C., followed by 0.3 volumes of a 300 mM aminoacid (Leu or Gly) solution in 300 mM NaOH for 2 hrs at 25° C. The totalvolume of the reactions was 200 uL. The crude linked amino acid reagentswere isolated by EtOH precipitation and used without furtherpurification. DNA was precipitated by NaOAc/EtOH according to (Sambrook,J., Fritsch, E. F. and Maniatis, T. (1989) in “Molecular Cloning: aLaboratory Manual”, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring, Harbor, N.Y.). The pellet was resuspended in water.

The oligonucleotides were annealed by preparing: 3.125 uM of eacholigonucleotides, 125 mM MES pH 6.0, 187.5 mM NaCl. The mixtures wereincubated at 80 degrees C. for 2 minutes followed by slow cooling toroom temperature in a water bath. The DNA directed chemical reaction(amide bond formation) were performed by adding EDC and sNHS to thepre-annealed oligonucleotedes; 2.5 uM preformed star structures, 100 mMMES pH 6.0, 150 mM NaCl, 20 mM EDC and 15 mM sNHS (finalconcentrations). The mixture was incubated overnight at room temperatureand EtOH precipitated as described above.

The reactions were analyzed by PAGE; both native (7.5% polyacrylamide),as well as by non-native PAGE (7.5% polyacrylamide, 8M urea), followedby staining with ethidium bromide, using standard protocols (Sambrook,J., Fritsch, E. F. and Maniatis, T. (1989) in “Molecular Cloning: aLaboratory Manual”, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring, Harbor, N.Y.).

The oligonucleotide, vip017, was functionalized by cross-linking a aminoacid, Leu or Gly, through the alfa-amine to the primary amine on amodified internal dT in vip017, by the homobifuntional linker BSOCOES.The modified dT is positioned between the two hybridization segments invip017. Vip076 has a modified dT containing a primary amine followed bya 3′ hybridization segment complementary to the 5′ hybridization segmentin vip017. Consequently, by annealing vip017 and vip076 proximity iscreated between the amino acid conjugated to vip017 and the primaryamine on vip076. Accordingly, a band corresponding to a dimer wasobserved in lane 4-6 in the native gel. Vip008 has complementarysegments to both vip076 and vip017 thus capable of forming a closedtrimer star structure and arranging the chemical functionalities ofvip017 and vip076 in the reaction chamber in the center of the starstructure. Accordingly, a band corresponding to a trimer was observed inlanes 1-3 in the native gel.

Upon activation by EDC/sNHS an amide bond can be formed between theamino acid conjugated to vip017 and the primary amine in vip076, andthereby cross-linking vip017 and vip076. As expected when starstructures were formed (vip008 present), a unique band corresponding tocross-linked vip076/vip017 did appear both with vip017-Gly andvip017-Leu (none-native PAGE, lanes 2 and 3, respectively), which wasnot present without EDC/sNHS activation (none-native PAGE, lanes 8 and9) or with non-acetylated vip017 (non-native PAGE, lane 1).Interestingly, the unique band was not detectable when vip008 was notpresent (non-native PAGE, lanes 5 and 6). This illustrates that achemical reaction can be directed by a star structure and that the starstructure seems more efficient in guiding chemical reactions than twoannealed oligoes.

Example 6 Assembling and Ligation of a Star Structure

The successful amplification of trimeric DNA star structure consistingof dsDNA was demonstrated in this example. Mutual complementarybi-specific oligonucleotides were annealed, ligated and subsequentlyused as a template in a PCR reaction. The following oligonucleotideswere used: vip029-vip031-vip0132-vip0133-vip030. FIG. 13 showsschematically the hybridization of the oligonucleotides.

DNA oligonucleotides (prepared by DNA Technology Århus, Denmark) weremixed in 2 μM concentrations each in 1× Ligase Buffer (New EnglandBiolabs), 50 mM NaCl. The mixtures were incubated as follows: 94° C. for5 minutes, 80° C. for 30 seconds, 65° C. for 30 seconds, 50° C. for 30seconds, 35° C. for 30 seconds, 20° C. for 30 seconds, 10° C. until nextstep. The annealing procedure was performed on an Applied BiosystemsAB2720 PCR machine.

The 5′ termini of the oligonucleotides were phosphorylated by T4 DNApolynucleotide kinase. A mixture consisting of 1.5 μM star structure,1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl and 0.17 U/μl T4DNA polynucleotide kinase (New England Biolabs, cat# M0201), wasprepared and incubated for 30 minutes at 37° C.

A phosphodiester bond between juxtaposed ends of annealedoligonucleotides was formed by T4 DNA ligase (New England Biolabs, cat#M0202), in 1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl, and200 U T4 DNA ligase (New England Biolabs, cat# M0202) in a volume of 10μl and incubated overnight at 16° C.

PCR amplification was performed using the following conditions:

Reactions were performed in 1× ThermoPol buffer (New England BiolabsB9004S), with 0.2 mM dNTPs (New England Biolabs O447S), 8 mM MgSO4, 0.2μM sense primer and 0.2 μM antisense primer, 1 M Betaine (Sigma B0300),1 U/100 μl of Vent (exo-) (New England Biolabs M0257L). Primers usedwere vip027 and vip028. Biotinylated versions of these two primers arevip034 and vip038, respectively. PCR amplification was performed usingthe following cycling conditions: 2 minutes at 95° C., and 20 cycles of95° C./30 sec, 60° C./30 sec, 74° C./30 sec. PCR product to be used forfolding and ssDNA purification reported in example 7 below, was madewith vip034 and vip028 primers. The PCR product was analyzed by nativePAGE. A band of 201 bp is clearly seen on the gel depicted on FIG. 13.

Example 7 Re-Folding of PCR Product

Folding of PCR products were performed as follows. A PCR cleanupprocedure was performed using PerfectPrep (Eppendorf, cat#0032 007.740)according to kit instructions. Folding was performed in 0.1 M NaCl, 0.1%Triton X-100, 0.1 μM vip027, 0.1 μM vip028, in a volume of 10 μl (5 μlper product mixed with 5 μl 2× buffer/primer mix). The PCR product wasused in 4 different concentrations (ranging from 1:2 dilution to 1:20dilution). In one series, the mixture was incubated for 2 minutes inboiling water, and subsequently cooled in an ice/water bath. In thesecond series, the mixture was heated and cooled using the followingprogram on an ABI2720 PCR machine: 94° C. for 5 minutes, 80° C. for 30seconds, 65° C. for 30 seconds, 50° C. for 30 seconds, 35° C. for 30seconds, 20° C. for 30 seconds, 10° C. until next step. In the thirdseries, no heating or cooling was performed.

The products were analyzed on 20% TBE urea gel (Invitrogen). The gel wasstained with SYBR green (Molecular Probes 57563, 1:10.000 dilution in1×TBE buffer, according to instructions).

In FIG. 14 the lanes of the gel comprise the following content:

Lane 1: PCR product diluted 1:2, quick cool

Lane 2: PCR product diluted 1:4, quick cool

Lane 3: PCR product diluted 1:10, quick cool

Lane 4: PCR product diluted 1:20, quick cool

Lane 5: PCR product diluted 1:2, step cool

Lane 6: PCR product diluted 1:4, step cool

Lane 7: PCR product diluted 1:10, step cool

Lane 8: PCR product diluted 1:20, step cool

Lane 9: PCR product diluted 1:2, no treatment

Lane 10: PCR product diluted 1:4, no treatment

Lane 11: PCR product diluted 1:10, no treatment

Lane 12: PCR product diluted 1:20, no treatment

Lane 13: PCR product only

The experiment shows that single stranded star structure DNA ismigrating at app. 1000 bp, in contrast to the 201 bp dsDNA product. Inappears that the optimal condition for star structure formation isheating followed by quick cooling of the reactions.

Example 8 Purification of Re-Folded Star Structure

The ssDNA star structure was purified using streptavidin-coated magneticbeads (Dynal, Cat#650.02). 10 pa beads were washed two times with 2×BWT(2 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100). Afterthe final wash, the beads were suspended in one volume 2×BWT and addedone volume refolded PCR product. The suspension was incubated for 15minutes at room temperature, mixing every once in a while. The tube wasplaced in the magnet, and after the beads had been collected thesupernatant was removed. The beads were suspended in 50° C. warm washbuffer (2 M urea, 0.1% Triton X-100), and incubated for 2 minutes at 50°C. The tube was placed on the magnet, and the wash procedure wasperformed three times in total. One final wash was performed in 1×BWT (1M NaCl, 5 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.1% Triton X-100).Following removal of the final wash buffer, the beads were suspended in1.5 ml NT (10 mM NaCl, 0.1% Triton X-100), and incubated at 50° C. for 5minutes. The tube was transferred to an ice/water bath for rapidcooling, and this procedure resulted in formation of the star structureimmobilized on the streptavidin-coated magnetic beads. The tube wasafter the rapid cooling placed in the magnet, and the supernatant wasremoved. The beads were suspended in 50 μl NT, ready for digest withBsaI for release of the ssDNA from the beads.

17 μl beads were added 2 μl 10×NEB3 buffer and 1 μl BsaI (10 U/μl; NEBR0535L). The reaction was incubated 1½ h at 50° C. The digest wasanalyzed by denaturing polyacrylamide gel electrophoresis (10% TBE-ureagel, Invitrogen), by adding denaturing loading buffer to the samples,and loading the whole mixture including the beads in the wells of thegel. The gel was stained with SYBR green (Molecular Probes S7563,1:10.000 dilution in 1×TBE buffer, according to instructions).

On FIG. 15 one band is observed in lane 1 (+BsaI) lane and no band isseen in lane 2 ‘-BsaI’ lane. Thus, the ssDNA was folded on thestreptavidin-coated magnetic beads thus forming the substrate for BsaIdemonstrated by the ability of the enzyme to cleave off the singlestranded product.

Example 9 Digest Loop Format

Two genomes were designed, both enabling digest in the loops of the starstructures. Restriction enzymes are in general not capable of digestingssDNA. However, annealing of 10-mer oligonucleotides to the loopsgenerates substrates for the enzymes, and thereby the recognitionsequences for the enzymes will become double-stranded. The design of theexperiment is schematically shown on FIG. 16.

Two structures were assembled, s129 and s149. s129 was composed of thefollowing oligonucleotides: vip029-vip161-vip162-vip163-vip070. s149 wascomposed of the following oligonucleotides:vip029-vip161-vip192-vip193-vip070. Vip162, vip163, vip 192 and vip 193all contain recognition sequences for two restriction enzymes (vip 162:ApaI and BamHI, vip163: EcoRI and KpnI, vip192: PvuII and SacL vip193:SmaI and VspI)

DNA oligoes (prepared by TAGC, Copenhagen, Denmark) were mixed in 2 μMconcentrations each in 1× Ligase Buffer (New England Biolabs), 50 mMNaCl. The mixtures were incubated as follows: 94° C. for 5 minutes, 80°C. for 30 seconds, 65° C. for 30 seconds, 50° C. for 30 seconds, 35° C.for 30 seconds, 20° C. for 30 seconds, 10° C. until next step. Theannealing procedure was performed on an Applied Biosystems AB2720 PCRmachine.

The 5′ termini of the oligonucleotides were phosphorylated by T4 DNApolynucleotide kinase. A mixture consisting of 1.5 μM star structure,1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl and 0.17 U/μl T4DNA polynucleotide kinase (New England Biolabs, cat# M0201), wasprepared and incubated for 30 minutes at 37° C.

A phosphodiester bond between juxtaposed ends of annealedoligonucleotides was formed by T4 DNA ligase (New England Biolabs, cat#M0202), in 1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl, and200 U T4 DNA ligase (New England Biolabs, cat# M0202) in a volume of 10μl and incubated overnight at 16° C.

PCR amplification was performed using the following conditions:

Reactions were performed in 1× ThermoPol buffer (NEB B9004S), with 0.2mM dNTPs (NEB O447S), 8 mM MgSO4, 0.2 μM sense primer and 0.2 μMantisense primer, 1 M Betaine (Sigma B0300), 1 U/100 μl of Vent (exo-)(NEB M0257L). Primers used were vip027 and vip028. Biotinylated versionsof the primers are vip034 and vip038, respectively. PCR amplificationwas performed using the following cycling conditions: 2 minutes at 95°C., and 20 cycles of 95° C./30 sec, 60° C./30 sec, 74° C./30 sec.

The result of the experiment is shown in FIG. 17.

In lane 1 and 3 it is seen that both genomes were amplifiedsuccessfully. Lanes 2 and 4 are negative controls without ligase addedfor the two genomes.

Purification of ssDNA structures of s129 and s149.

80 μl PCR product (made using vip034 and vip028 primers) of each genomewas purified using PerfectPrep (Eppendorf, cat#0032 007.740) accordingto kit instructions. Elution was done in 40 μl elution buffer. Forrefolding of the PCR products, the following was performed: to 40 μl PCRproduct, add 750 μl 0.2 M NaCl, 0.2% Triton X-100, 7.5 μl vip027 and 7.5μl vip028 and 695 μl H2O. Incubate the mix in boiling water for 5minutes, and cool quickly in an ice-water bath.

20 μl Streptavidin beads (Dynal, 650.02) were washed two times with2×BWT (2 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100).After the final wash, the beads were suspended in one volume 2×BWT andadded one volume refolded PCR product. The suspension was incubated for15 minutes at room temperature, mixing every once in a while. The tubewas placed in the magnet, and after the beads had been collected thesupernatant was removed. The beads were suspended in 50° C. warm washbuffer (2 M urea, 0.1% Triton X-100), and incubated for 2 minutes at 50°C. The tube was placed on the magnet, and the wash procedure wasperformed three times in total. One final wash was performed in 1×BWT (1M NaCl, 5 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.1% Triton X-100). Afterseparation on the magnet, the beads were suspended in 50 μl 10 mM NaCl,0.1% Triton X-100.

20 μl of the beads of each prep were separated on the magnet, andsuspended in 10 μl 100 mM NaCl, 0.1% Triton X-100. Each preparation wassplit into two tubes of 5 μl each, and digest oligoes were added asfollows:

1: s129-vip164-ApaI

2: s129-vip165-KpnI

3: s149-vip194-PvuII

4: s149-vip195-VspI

0.25 μl oligo (20 μM stock) was added, resulting in a final conc. of 1μM. Annealing was performed on AB2720 PCR machine using the program: 50°C.-2 minutes; 40° C.-2 minutes; 35° C.-2 minutes; 30° C.-2 minutes; 30°C.-2 minutes; 25° C.-2 minutes; 20° C.-2 minutes.

The beads were washed in 100 μl 100 mM NaCl, 0.1% Triton X-100. Thebeads were subsequently suspended in 18 μl 1.1× digest buffer. Thereactions were split into two tubes, 9 μl each, and 1 μl enzyme (10Units) were added to each tube, resulting in 1× digest buffer for thedigest. One set of digests was incubated at 30° C. and the other set wasincubated at 37° C. Incubation was done for 5 h, mixing every once in awhile to suspend the beads from the bottom of the tubes.

Following digest, the products were analyzed on 10% TBE-urea gel(Invitrogen). Loading buffer was added to the digests, and the wholereaction mix including the beads was loaded on the gel. The gel wasstained with SYBR green (Molecular Probes S7563, 1:10.000 dilution in1×TBE buffer, according to instructions).

The result of the experiment is shown in FIG. 18.

Lane 1 shows ApaI digest of s129 at 37° C.

Lane 2 shows KpnI digest of s129 at 37° C.

Lane 3 shows PvuII digest of s149 at 37° C.

Lane 4 shows VspI digest of s149 at 37° C.

Lane 5 shows ApaI digest of s129 at 30° C.

Lane 6 shows KpnI digest of s129 at 30° C.

Lane 7 shows PvuII digest of s149 at 30° C.

Lane 8 shows VspI digest of s149 at 37° C.

Expected Band Sizes are as Follows:

ApaI: 163 nt, KpnI: 80 nt, PvuII: 165 nt, VspI: 83 nt.

On the gel, bands of the expected sizes do appear for all four enzymes.Note that the fragments containing biotin will not enter the gel as theyare bound to the beads. Both temperatures give a product, showing therobustness of the procedure.

Example 10 DNA Directed Formation of Amide Bonds in Various Topologies

The oligonucleotide, vip017, was functionalized by cross-linking anamino acid (Glycine, Fluka cat #50052) through the alfa-amine to theprimary amine on an internal modified dT in vip017, by thehomobifuntional linker BSOCOES((Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), Pierce cat#21600) bytreatment of the oligonucleotide (5 nmol) in a 200 mM pH 7.4 sodiumphosphate solution (200 uL) with 0.1 volumes of a 100 mM BSOCOESsolution in DMF for 10 min at 25° C., followed by 0.3 volumes of a 300mM amino acid (Glycine) solution in 300 mM NaOH for 2 hrs at 25° C. Thetotal volume of the reaction was 200 uL. The crude linked amino acidreagents were isolated by EtOH precipitation and used without furtherpurification.

DNA was precipitated by NaOAc/EtOH according to (Sambrook, J., Fritsch,E. F. and Maniatis, T. (1989) in “Molecular Cloning: a LaboratoryManual”, Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring, Harbor, N.Y.). The pellet was resuspended in water.

The oligonucleotides were annealed by preparing: 0.556 uM of eacholigonucleotide, 111 mM MOPS pH 6.5 (Fluka 69947), 1.11 M NaCl (Fluka71376), and subsequently incubated as follows: 94° C. for 5 minutes, 80°C. for 30 seconds, 65° C. for 30 seconds, 50° C. for 30 seconds, 35° C.for 30 seconds, 20° C. for 30 seconds, 10° C. until next step. Theannealing procedure was performed on an Applied Biosystems AB2720 PCRmachine.

The DNA directed chemical reaction (amide bond formation) were performedby adding 100 mM DMTMM (Acros, cat #A017657001) to the pre-annealedoligonucleotides. DMTMM was dissolved in water at a concentration of 1M. Before adding DMTMM, the reaction mixtures were preheated to 50° C.Final concentration of each oligonucleotide was 0.5 μM. The reactionswere performed in 100 mM MOPS pH 6.5, 1 M NaCl, 100 mM DMTMM (finalconcentrations), in a volume of 20 μl, at 50° C. for 3 h.

The reactions were analyzed by PAGE; both native (20% polyacrylamide,Invitrogen), and by denaturing PAGE (10% polyacrylamide, 7 M urea,Invitrogen), followed by staining with SYBR green (Molecular Probes57563, 1:10.000 dilution in 1×TBE buffer, according to instructions).All results are shown in FIG. 19.

The oligonucleotide, vip017, was functionalized by cross-linking anamino acid (Glycine) through the alfa-amine to the primary amine on amodified internal dT in vip017, by the homobifuntional linker BSOCOES.The modified dT is positioned between the two hybridization segments invip017. Vip008 does not contain an acceptor amine on a modified dT, itcontains one hybridization segment with which it will hybridize toVip017, and thus it will not be covalently cross-linked to Vip017-Glyupon the addition of DMTMM. Accordingly, no visible band was observed inlane 1.

Vip018-NH2 has a modified dT containing a primary amine followed by a 3′hybridization segment complementary to the 5′ hybridization segment invip017. Consequently, by annealing vip017-Gly and vip018-NH2 proximityis created between the amino acid conjugated to vip017 and the primaryamine on vip018. Accordingly, a band corresponding to a dimer composedof cross-linked vip017-Gly/vip018-NH2 was observed in lane 2.

Vip006 has complementary segments to both vip017-Gly and vip018-NH2, andit is thus capable of forming a closed trimer star structure andarranging the chemical functionalities of vip017-Gly and vip018-NH2 inthe reaction chamber in the center of the star structure. Accordingly, aband corresponding to a dimer composed of cross-linkedvip017-Gly/vip018-NH2 was observed in lane 3. Furthermore, lane 3contained more stain than lane 2, indicating that the closed trimer starstructure creates better reaction conditions than an open dimerstructure does.

Vip020-N112 has a modified dT containing a primary amine, but itcontains no hybridization segments to vip017-Gly. Consequently, no bandsappear in lane 4 because the reactants of vip020-NH2 and vip017-Gly arenot brought into proximity by base airing.

Vip006 has one hybridization segment capable of hybridizing to a segmentof vip017-Gly, and another hybridization segment capable of hybridizingto vip020-NH2, which has a modified dT containing a primary aminebetween its two hybridization segments. Thus, vip006 brings thefunctional groups into proximity, and a band is visible on in lane 5,constituting cross-linked vip017-Gly and vip020-NH2.

Vip009 has one hybridization segment capable of hybridizing to a segmentof vip017-Gly, and another hybridization segment capable of hybridizingto vip020-NH2, which has a modified dT containing a primary aminebetween its two hybridization segments. Thus, vip009 brings thefunctional groups into proximity. Accordingly, a band was observed inlane 6, constituting cross-linked vip017-Gly and vip020-NH2.

Vip006 has one hybridization segment capable of hybridizing to a segmentof vip017-Gly, and another hybridization segment capable of hybridizingto vip020-NH2, which has a modified dT containing a primary aminebetween its two hybridization segments. Vip009 has one hybridizationsegment capable of hybridizing to a segment of vip017-Gly, and anotherhybridization segment capable of hybridizing to vip020-NH2, which has amodified dT containing a primary amine between its two hybridizationsegments. Hybridizing these four oligonucleotides result in theformation of a tetramer star structure. In lane 7 a band was observed,showing that the functional groups on vip017-Gly and vip020 are broughtinto proximity of each other by the hybridization of the fouroligonucleotides. Furthermore, lane 7 contained more stain than bothlane 5 and 6, indicating that the closed tetrameric star structurecreates better reaction conditions than open trimer structures do.

Vip048-NH2 has a modified dT containing a primary amine and onehybridization segment capable of binding to vip017-Gly. Between themodified dT with the primary amine and the hybridization segment capableof annealing to vip017-Gly is inserted 6 nucleotides not involved inhybridization (wobble nucleotides). A band was observed in lane 8, thusindicating cross-linking of the two oligonucleotides. However, the 6extra nucleotides introduced did lower the amount of cross-linkedproduct obtained as seen by comparing lane 8 and lane 2.

Vip006 has one hybridization segment capable of hybridizing to a segmentof vip017-Gly, and another hybridization segment capable of hybridizingto vip048. Vip048-NH2 has a modified dT containing a primary amine andone hybridization segment capable of binding to vip017-Gly. Between themodified dT with the primary amine and the hybridization segment capableof annealing to vip017-Gly is inserted 6 nucleotides not involved inhybridization (wobble nucleotides).

The presence of vip006 brings the two reactive groups into proximity,and a product is formed as seen in lane 9. The band intensity wasstronger than that seen in lane 8, indicating that vip006 stabilizes thestructure (trimer star structure) and the reaction proceeds better thanthat found in the dimer format of the reaction.

Vip048-NH2 has a modified dT containing a primary amine and onehybridization segment capable of binding to vip017-Gly. Between themodified dT with the primary amine and the hybridization segment capableof annealing to vip017-Gly is inserted 6 nucleotides not involved inhybridization (wobble nucleotides). Vip056 contains one hybridizationsegment capable of hybridizing to vip017-Gly, and another hybridizationsegment capable of hybridizing to the segment of vip048-NH2 containingthe 6 wobble-nucleotides. Thus, upon annealing of these threeoligonucleotides, the primary amine on the modified dT on vip048 ismoved 6 nucleotides away for the reaction chamber and are now located inthe double stranded arm. Double stranded DNA is in contrast to singlestranded DNA very rigid thus preventing the conjugated moiety to movefreely and thereby decreasing its reactivity. Accordingly, only a veryfaint stain was observed in lane 10.

Vip018-NH2 has a modified dT containing a primary amine followed by a 3′hybridization segment complementary to the 5′ hybridization segment invip017-Gly.

Vip056 contains one hybridization segment capable of hybridizing tovip017-Gly, and another hybridization segment capable of hybridizing tothe segment of vip018-NH2. Between the two hybridization segments,vip056 contains 6 extra nucleotides (wobble nucleotides). A dimer bandis seen in lane 11, indicating that the reactive groups are brought intoproximity of each other for a chemical reaction to occur, and the 6wobble nucleotides in vip056 does not impair the proximity with thecurrent architecture in this experiment.

Example 11 Chemical Preparation of Various Oligonucleotides Example 11.1Acetylation of an Oligonucleotide Having Internal Modified dT(Amine-C6-dT) (Position n=1)

Acetylation with Fmoc-AA-OH promoted by DMT-MM.

The oligonucleotide Vip068 having an internal amine-C6-dT, was acylatedwith Fmoc-Leu-OH promoted by DMT-MM(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,Fluka #74104) by treatment of the oligonucleotide (500 pmol) dissolvedin a 1:1 mixture of DMF and 300 mM NaCl together with one of thefollowing buffers: sodium phosphate 400 mM, pH 7.0, MOPS 400 mM, pH 7.5,HEPES 400 mM pH 8.0, sodium phosphate 400 mM, pH 8.8, with DMT-MM 50 mM.Total reaction volume was 20 μL. Reactions were incubated 16 hrs at 25°C. The reaction mixture was diluted to 50 μL and purified on a spincolumn (Amersham Biosciences #27-5325-01) according to manufacturesprotocol followed by purification by HPLC and mass spectrometryanalysis.

General Purification Method:

Functionalized oligonucleotides were purified by a Hewlett PackardAgilent HPLC instrument with auto sampler and fraction collector on anXTerra C18 column (Waters #186000602) using acetonitrile/TEAA 100 mM pH7.0 mixtures as eluent. Appropriate fractions were lyophilized anddiluted to 20 mM with water.

General Mass Spectrometry Analysis:

Functionalized oligonucleotides were analyzed by MALDI-TOF massspectrometry on a Bruker AutoFlex instrument in a HPA/ammonium citratematrix using negative ion reflector mode.

DNA Calculated mass Found mass vip068-LeuFmoc 6843.340 6844.5

Example 11.2 Acetylation of an Oligonucleotide Having Internal ModifieddT (Amine-C6-dT) (Position n=1)

Acetylation with Fmoc-AA-OSu

The oligonucleotide Vip046 was acylated with Fmoc-AA-OSu (AA=Gly or Leu)by treatment of the oligonucleotide (125 pmol) dissolved in a 1:1mixture of DMF and sodium phosphate buffer 100 mM, pH 7.4 with 25 mMFmoc-Gly-OSu (Chemlmpex #02420) or Fmoc-Leu-OSu (ChemImpex #02429) for 2hrs at 25° C. The functionalized oligonucleotide was precipitated byNH4OAc/EtOH according to (Sambrook, J., Fritsch, E. F. and Maniatis, T.(1989) in “Molecular Cloning: a Laboratory Manual”, Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring, Harbor, N.Y.). The pelletwas resuspended in water and analyzed by MALDI-TOF mass spectrometry.

DNA Calculated mass Found mass vip046-LeuFmoc 6932.3642 6932.1vip046-GlyFmoc 6876.3016 6876.1

Example 11.3 Acetylation of Oligonucleotides Having Internal Modified dT(Amine-C6-dT) (Position n=1)

Synthesis of peptide-oligonucleotide conjugates (single letterabbreviation used for amino acid): YGGFL-Vip068, GLFYG-Vip068,YGGFL-PEG-Vip068, GLFYG-PEG-Vip068, GFL-Vip016 and GFL-PEG-Vip016:Acetylation with Fmoc-AA-OSu with subsequent deprotection on Sepharose.

The peptides were synthesized from the primary amine on an internalmodified dT on the oligonucleotides absorbed on DEAE Sepharose (Sigma#DFF100). Amino acids were coupled by rounds of acylation withFmoc-AA-OSu (AA=Gly, Leu, Phe, Tyr, C6, PEG) (Fmoc-L-GlycineN-hydroxysuccinimide ester, Chemlmpex #02420; Fmoc-L-leucineN-hydroxysuccinimide ester, Chemlmpex #02429; Fmoc-L-phenylalanineN-hydroxysuccinimide ester, Chemlmpex #02446; Fmoc-L-tyrosineN-hydroxysuccinimide ester, Chemlmpex #11972; Fmoc-6-aminohexanoic acidN-hydroxysuccinimide ester, Chemlmpex #7296;Fmoc-8-amino-3,6-dioxaoctanoic acid hydroxysuccinimide ester,synthesized from Fmoc-8-amino-3,6-dioxaoctanoic acid (Chemlmpex #7310)by EDC coupling with N-hydroxysuccinimide) followed by Fmoc deprotectionaccording to the procedure of Halpin and Harbury (Plos Biology 2004, 2,1-8). After elution of the DNA from the sepharose the mixture wasdesalinated on a spin column (Amersham Biosciences #27-5325-01)according to manufactures protocol followed by purification by HPLC.Yields were determined by HPLC.

DNA Isolated yield YGGFL-Vip068 19% YGGFL-PEG-Vip068 32% GLFYG-Vip06811% GLFYG-PEG-Vip068 18% GFL-Vip016 30% GFL-PEG-Vip016 36%

Example 11.4 Acetylation of Oligonucleotides Having Internal Modified dT(Amine-C6-dT) (Position n=1)

Synthesis of acceptor oligonucleotide with a C6-NH2, PEG-NH2 and Gly-NH2linker.

Oligonucleotide, vip016, absorbed on DEAE Sepharose (Sigma #DFF100) wasacylated at the primary amine on an internal modified dT in vip016 withFmocNH-C6-CO2Su (Fmoc-6-aminohexanoic acid N-hydroxysuccinimide ester,ChemImpex #7296), FmocNH-PEG-OSu (Fmoc-8-amino-3,6-dioxaoctanoic acidhydroxysuccinimide ester, synthesized fromFmoc-8-amino-3,6-dioxaoctanoic acid (Chemlmpex #7310) by EDC couplingwith N-hydroxysuccinimide) or Fmoc-Gly-OSu (Fmoc-L-GlycineN-hydroxysuccinimide ester, ChemImpex #02420) followed by cleavage ofthe Fmoc protecting group according to the procedure of Halpin andHarbury (Plus Biology 2004, 2, 1-8) Reactions were performed on 1 nmolDNA. After elution of the DNA from the sepharose the mixture wasdesalted on a spin column (Amersham Biosciences #27-5325-01) accordingto manufactures protocol followed by purification by HPLC. Yields weredetermined by HPLC.

DNA Isolated yield H2N-PEG-vip016 39% H2N-C6-vip016 39% H-G-vip016 45%

Example 11.5 Conjugation of Amino Acids to Oligonucleotides HavingInternal Modified dT (Amine-C6-dT) via BSOCOES or DSS at Position n≠1

The oligonucleotides, vip046, vip017, vip047 and vip048, werefunctionalized by cross-linking an amino acid (Gly, L-Leu, L-Phe, L-Tyr,Fluka, #50052, #61820, #78020, #93829) through the alfa-amine to theprimary amine on an internal modified dT in the oligonucleotide, by thehomobifunctional linkers BSOCOES((Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), Pierce cat#21600) andDSS (Disuccinimidyl suberate, Pierce #21655) by treatment of theoligonucleotide (0.5 nmol) and the amino acid (15 mM) in a 1:1 mixtureof DMF and 400 mM pH 8.4 sodium phosphate buffer with the linker (10mM). Total volume of the reactions was 20 μL. The reactions wereincubated 4 hrs at 25° C. The reaction mixture was diluted to 50 μL andpurified on a spin column (Amersham Biosciences #27-5325-01) accordingto manufactures protocol followed by purification by HPLC. Yields weredetermined by HPLC. Identity was determined by MALDI-TOF massspectrometry.

DNA Isolated yield Calculated mass Found mass vip046-BSOCOES-Gly 32%6878.2325 6879.3 vip046-BSOCOES-Leu 48% 6934.2951 6936.1vip046-BSOCOES-Phe 49% 6968.2795 6969.0 vip046-BSOCOES-Tyr 51% 6984.27446985.5

Example 12 Stability of Acylation Reaction in Reaction Center atDifferent Temperatures

In this example it is demonstrated how transfer of one amino acid can beeffected at elevated temperatures in a trimer star structure. Resultsare shown in FIG. 20.

Oligos vip006, vip018, and vip017-Leu (DNA Technology Arhus, Denmark,vip017 derivatized as described in example 11 as 20 mM stock solutionswere mixed in buffer solution containing a final composition ofmorpholinopropanesulfonic acid (MOPS, 100 mM, pH 7.0) and NaCl (1M).Solutions were subjected to an annealing program (PCR machine: 5 min @94° C., 30 sec @ 80° C., 30 sec @ 65° C., 30 sec @ 50° C., 30 sec @ 35°C., 30 sec @ 20° C., 30 sec @ 10° C.). Each reaction was added chemicalactivator (DMTMM, Fluka #74104, 100 mM aq. sol, final concentration of 5mM) and incubated for indicated time at 25° C. or 70° C.

Reaction mixtures were analyzed by denaturing (10%) PAGE and bands werevisualized by SYBR Green stain (fixation of gel in 50% EtOH for 5 min,wash in water bath 5 min, then incubated for 10 min in 10.000 folddiluted DMSO stock of SYBR Green in 1×TBE buffer).

For reactions run at 25° C., an indetectable amount of product wasobserved after the first 2 h (reactions 1-5). In the 4 h reaction(reaction 6), a small amount was observed. Significant amounts wereobserved after an over night incubation with highest intensity observedwhere more activator was added.

For reactions run at 70° C., the first trace of product was observedafter 5 min (reaction 2). Increasing amounts were observed up to 240min, then decreasing amounts for over night reactions.

The desired cross-linked product of vip017-Leu and vip018 was clearlyformed at both 25° C. and 70° C., thus demonstrating that starstructures are able to mediate reactions run at elevated temperatures.The rate of reaction is clearly different at the two temperatures usedin this experiment. However, this would be expected as the initialactivation of the amino acid carboxylic acid by DMTMM is controlled byintermolecular collisions only, not dirigation by DNA.

Example 13 Stability of Acylation Reaction in Reaction Center atDifferent pH's

In this example it is demonstrated how one amino acid can be transferredin a trimer star structure in a pH span of 5.2 to 8.0. At low pH, theacceptor amino group may be partially protonated, thus inactivating itas a potent nucleophile. At higher pH, reactive intermediates producedby activation of the amino acid carboxylic acid can be hydrolyzed, thusdeactivating it and destroying the chemical activator. Both may hamperformation of the desired amide bond. Results are shown in FIG. 21.

Oligos vip016, vip008, and vip017-Tyr (DNA Technology Arhus, Denmark,vip017 derivatized as described in example 11) as 20 mM stock solutionswere mixed in 2M NaCl and subjected to an annealing program (PCRmachine: 5 min @ 94° C., 30 sec @ 80° C., 30 sec @ 65° C., 30 sec @ 50°C., 30 sec @ 35° C., 30 sec @ 20° C., 30 sec @ 10° C.). This annealingmixture was diluted into buffer solutions to a final composition ofmorpholinopropanesulfonic acid (MOPS, 100 mM, pH 5.2, 5.5, 6.0, 6.5,7.0, 7.5, or 8.0), NaCl (1M) and chemical activator (DMTMM, Fluka#74104, 1.0M aq. sol, final concentration of 75 mM) and incubated for 1h at 50° C. Final DNA concentration was 0.5 mM. Reaction mixtures wereanalyzed by denaturing PAGE (10% gel), which was incubated with SYBRgreen (10.000 dilution in TBE-EtOH (96%) 1:1 from DMSO stock) for 10min.

All seven lanes covering pH between 5.2 and 8.0 showed a strong band forthe cross-linked oligos vip016-vip017-Tyr indicating a broad pH windowin which to operate. Thus, protonation of acceptor amine or hydrolysisof reactive intermediates is not a significant problem using the aboveconditions.

Example 14 Stability of Acylation Reaction in Reaction Center atDifferent Levels of Organic Solvent

To demonstrate the stability of DNA star structures, the transfer of oneamino acid was carried out in a mixture of H₂O and an organic solvent,thus resembling conditions used in organic chemical synthesis. If basepairing and star structure was destroyed under these conditions, nocross linked product can be formed. The solvents dioxan, acetonitrile,and tetrahydrofuran were chosen with regard to miscibility with waterand general applicability in organic synthesis.

Oligos vip016, vip008, and vip017-Phe (DNA Technology Arhus, Denmark,vip017 derivatized as described in example 11) as 20 μM stock solutionswere mixed in a buffer of MOPS (200 mM, pH 6.5; Fluka #69947) and NaCl(2M; Fluka #71376), and subjected to an annealing program (PCR machine:5 min @ 94° C., 30 sec @ 80° C., 30 sec @ 65° C., 30 sec @ 50° C., 30sec @ 35° C., 30 sec @ 20° C., 30 sec @ 10° C.). This annealing mixturewas diluted into mixtures of solvent and water to a final composition ofmorpholinopropanesulfonic acid (MOPS, 100 mM, pH 6.5), NaCl (1M),solvent (0, 10, 20, 30, or 35 vol %), and chemical activator (DMTMM,Fluka #74104, 1.0M aq. sol, final concentration of 75 mM), which wasincubated at 50° C. for 1 h. Final DNA concentration was 0.5 μM.Reaction mixtures were analyzed by denaturing PAGE (10% gel), which wasstained with SYBR green (Molecular Probes, #S7563) according tomanufactures instructions. The results are shown in FIG. 22

For dioxan, cross linked product was formed with up to 20% solvent. Onthe other hand, for all reactions containing acetonitrile ortetrahydrofuran similar amounts of product was formed, thus indicatingthat the presence of at least up to 35% of the organic solvent was welltolerated and DNA base paring was intact.

Example 15 Stability of Reaction Center at Different Levels of DMF

To demonstrate the stability of DNA star structures, the transfer of oneamino acid was carried out in a H2O-DMF mixture, thus resemblingconditions used in organic chemical synthesis. If base pairing and starstructure was destroyed under these conditions, no cross linked productcan be formed.

Oligos vip016, vip008, and vip017-Phe (DNA Technology Arhus, Denmark,vip017 derivatized as described in example 11) as 20 μM stock solutionswere mixed in a buffer of MOPS (500 mM, pH 6.5; Fluka #69947) and NaCl(4M; Fluka #71376), and subjected to an annealing program (PCR machine:5 mM @ 94° C., 30 sec @ 80° C., 30 sec @ 65° C., 30 sec @ 50° C., 30 sec@ 35° C., 30 sec @ 20° C., 30 sec @ 10° C.). This annealing mixture wasdiluted into mixtures of DMF and water to a final composition ofmorpholinopropanesulfonic acid (MOPS, 12.5 mM, pH 6.5), NaCl (100 mM),DMF (0, 10, 20, 30, 40, 50, 60, or 70 vol % DMF), and chemical activator(DMTMM, Fluka #74104, 1.0M aq. sol, final concentration of 75 mM), whichwas incubated over night at 25° C. Final DNA concentration was 0.5 μM.Reaction mixtures were analyzed by denaturing PAGE (10% gel), which wasstained with SYBR green (Molecular Probes, #S7563) according tomanufactures instructions. The results are shown in FIG. 23

The product band produced in the first five lanes were of similarintensity, thus indicating that the presence of at least up to 40% ofthe organic solvent, DMF, was well tolerated and DNA base paring wasintact. At 50% DMF, a weak band was still observed, but from 60% andabove no cross linked product was detected.

Example 16 Different Activators for Mediating Chemical Reaction

This example serves to illustrate how various chemical activators andauxiliary nucleophiles can be used to mediate the acylation of anacceptor amine with an amino acid. It is known from peptide chemistrythat addition of an axiliary nuclephile can greatly enhance the rate ofacylation and/or change the final outcome of the reaction. In thisexample reactions were nm using no axiliary nucleophile,N-hydroxysuccinimide (NHS, e.g. Fluka #56480), N-hydroxysulfosuccinimidesodium salt (s-NHS, Fluka #56485), or N-hydroxybenzotriazole hydrate(e.g. Fluka #54804) were used with DMTMM (Fluka #74104) or EDC (e.g.Fluka #03449) as activators.

Oligos vip016, vip008, and vip017-Tyr (DNA Technology Arhus, Denmark,vip017 derivatized as described in example 11) as 20 mM stock solutionswere mixed in a buffer of MOPS (200 mM, pH 6.5) and NaCl (2M), andsubjected to an annealing program (PCR machine: 5 min @ 94° C., 30 sec @80° C., 30 sec @ 65° C., 30 sec @ 50° C., 30 sec @ 35° C., 30 sec @ 20°C., 30 sec @ 10° C.). This annealing mixture was diluted with water andsolutions of additives to a final composition ofmorpholinopropanesulfonic acid (MOPS, 100 mM, pH 6.5), NaCl (1M),auxiliary nucleophile (final conc. 25 mM), and chemical activator (DMTMMor EDC, 0.5M aq. sol, final concentration of 75 mM). Final DNAconcentration was 0.5 mM. Reactions were incubated for 1 h at 50° C.

Reaction mixtures were analyzed by denaturing PAGE (10% gel), which wasincubated with SYBR green (10.000 dilution in TBE-EtOH (96%) 1:1 fromDMSO stock) for 10 min. Results are shown on FIG. 24.

Reactions 1-4 using DMTMM as activator, no difference was observed byusing additive or none. Most clearly was observed for EDC only, in whichcase no, or only faintly, product was observed. However, addition ofeither auxiliary nucleophile gave a comparable amount of cross linkedproduct again. This demonstrates that auxiliary nucleophile us neededwhen using EDC as activator and otherwise it is well tolerated in thereaction mixture.

Example 17 Transfer of Amino Acid to Other Acceptors

This example demonstrates how an amino acid can effectively betransferred to a number of acceptor amine linked in various ways. Vip016carrying a C6-NH2 served as control as before. Other acceptors weretripeptide GFL-vip016, PEG linked tripeptide GFL-PEG-vip016, aminofunctionalized NH2-PEG-vip016, amino functionalized NH2-C6-vip016, andglycine in G-vip016 (all prepared as described in example XX)

Oligos vip016-X-NH2, vip008, and vip017-Gly (DNA Technology Arhus,Denmark, vip017 derivatized as described in example XX) as 20 mM stocksolutions were mixed in a buffer of MOPS (200 mM, pH 6.5) and NaCl (2M),and subjected to an annealing program (PCR machine: 5 min @ 94° C., 30sec @ 80° C., 30 sec @ 65° C., 30 sec @ 50° C., 30 sec @ 35° C., 30 sec@ 20° C., 30 sec @ 10° C.). This annealing mixture was diluted withwater and solution of activator to a final composition ofmorpholinopropanesulfonic acid (MOPS, 100 mM, pH 6.5), NaCl (1M), andchemical activator (DMTMM, Fluka #74104, 1.0M aq. sol, finalconcentration of 75 mM). Final DNA concentration was 0.5 mM. Reactionswere incubated for 1 h at 50° C.

Reaction mixtures were analyzed by denaturing PAGE (10% gel), which wasincubated with SYBR green (10.000 dilution in TBE-EtOH (96%) 1:1 fromDMSO stock) for 10 min. Results are shown in FIG. 25.

All six reactions show a strong band from cross linked product. Lane 4and 5 were observed to run marginally slower compared to others becauseof larger peptide (tetrapeptide). Thus, transfer of one glycine to aminogroup linked via tripeptide +/−PEG linker, PEG linker itself, C6 linker,or glycine directly gives consistent results. This demonstratesrobustness in the reactor formed by star structures. Increasing size ofacceptor has little or no effect of the outcome of the crosslinking.

Example 18 Assembly and Subsequent Binding of Two Structural DNA DisplayProducts

Two Structural DNA display products were formed: Leu-enkephalin(Tyr-Gly-Gly-Phe-Leu-DNA) and scrambled Leu-enkephalin(Gly-Leu-Phe-Tyr-Gly-DNA). The latter was included as a negative controlfor the partitioning assay using the Leu-enkephalin specific monoclonalantibody 3E7. Key steps of the process are illustrated in FIG. 26.

DNA oligonucleotides were purchased by DNA Technology (Aarhus, Denmark)and functionalized as described in Example 11.

First step of the process involved annealing of the following oligoes,in two separate reactions to form Leu-Enkephalin-DNA and scrambleLeu-Enkephalin respectively. In reaction 1 (R1); Gly-Phe-Leu-PEG-vip231,Gly-BSOCOES-vip262 and vip088 (position 1, 2 and 3 in a 3-way DNA StarStructure, respectively) and in reaction 2 (R2): Gly-Tyr-Phe-PEG-vip238,Leu-BSOCOES-vip269 and vip088 (position 1, 2 and 3 in a 3-way DNA StarStructure, respectively). The three oligoes in the reactions willhybridize to each other, thus forming a three-way junction, where theattached amino acids are located in the centre of the structure. Notethat vip088 does not have a chemical functionality attached. Vip088'sfunction is simply to hybridize to the two other oligoes to form theclosed three-way junction.

200 pmoles of each oligo were mixed in 100 mM morpholinopropanesulfonicacid (MOPS, Fluka #69947) pH 6.5 and 1 M NaCl in a total volume of 370μl. The annealing of the oligoes was performed by incubation for fiveminutes at 95° C., before cooling to room temperature over approximately30 minutes. The activator DMT-MM (Fluka #74104) was dissolved in waterand added to a final concentration of 75 mM. The final reaction volumewas 400 μl. The chemical reaction was incubated for 1 hour at 50° C.Then, the product was ethanol precipitated by adding 2.5 volumes ethanoland 1 μl GenElute (Sigma 56575) to each reaction, and centrifuging thetubes 30 minutes, 20000×g at 4° C. The pellets were washed with 70%ethanol, before they were air-dried, and re-suspended in water.

Then, the samples were subjected to preparative non-nativepolyacrylamide gel electrophoresis (PAGE); 10% TBE-urea gel (Invitrogen)according to manufactures instructions. The band corresponding to thecross-linked product was cut out and the product extracted by the “crushand soak” method (Sambrook, J., Fritsch, E F, and Maniatis, T. (1989)in: Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory); the gel piece was crushed and soaked overnight in 400 TBEBuffer. The samples were ethanol precipitated as described above. Theprecipitates were dissolved in 1× Ligase buffer (New England Biolabs),50 mM NaCl. Then, the 5′ ends were phosphorylated by PolynucleotideKinase; 50 units Polynucleotide Kinase (NEB M0201) were include in atotal of 200 μl reaction volume. The reactions were incubated for 30 minat 37° C.

Then, the two cross-linked oligoes were transformed into a continuousDNA strand by a DNA ligase, which formed a phosphordiesther bond betweenthe juxtaposed 3′ end of vip231 and 5′ end of vip262 for R1 and thejuxtaposed 3′ end of vip238 and 5′ end of vip269 for R2. T4 DNA ligase(NEB M0202L) was added in 1× Ligase buffer, 50 mM NaCl, and 5 units/μlenzyme was added, giving a final reaction volume of 300 μl. The ligationwas incubated overnight at 16° C.

Then, the cleavable BSOCOES linker was eliminated.3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS, Fluka#29338) (pH 11.8)buffer and 2-mercaptoethanol (Fluka#63689) were added giving finalconcentrations of 100 and 60 mM, respectively, in a final reactionvolume of 600 μl. The reactions were incubated for 2 hours at 37° C. Thereactions were then neutralized by adding 200 μl 1 M MOPS buffer, pH6.5. Then, the DNA was ethanol precipitated according to the standardprocedure. After air drying of the pellets, the DNA was ready for thenext step, which was introduction of functionalized oligonucleotides onthe position 3. Note that by the elimination of the BSOCOES linker twoprimary amines are formed: one is at the terminus of the growing peptidechain on the original position 1 oligonucleotide and the other islocated on the original position 2 oligonucleotide. The so called“wobbling” strategy was used to favour a subsequent reaction between thegrowing peptide chain and the incoming amino acid on position 3 in thenext step: the oligoes on position 2 contain upstream of the modifiedbase a stretch of bases (wobbling bases), which are unpaired during thefirst chemical transfer, however in the second transfer process they arebase paired with the position 3 oligo. Consequently, the primary amineon the original position 2 oligonucleotide is now separated from thecentre of structure by a stretch of dsDNA which will decrease itsreactivity with moieties in the centre of the Star Structure becausedsDNA is rigid.

R1 product was annealed to Tyr-BSOCOES-vip263 and R2 product wasannealed to Gly-BSOCOES-vip270 under the following conditions: 200pmoles oligo, in 100 mM MOPS, pH 6.5, 1 M NaCl in a total volume of 240μl. The mixture was incubated 5 minutes at 95° C., before cooling toroom temperature over a period of approximately 30 minutes. Then, theactivator DMT-MM (Fluka #74104) was added to a final concentration of 75mM, for promoting the chemical reaction between the amino acids. Thechemical reaction was incubated for 1 hour at 50° C. The samples wereprecipitated by ethanol and subsequently subjected to preparativenon-native PAGE (as described above) where the band corresponding to thecross-linked product was excised from the gel. Then, each cross-linkedproduct were transformed into a continuous DNA strands by a DNA ligase,which formed a phosphordiesther bond between the juxtaposed 3′ end ofthe original vip232 and 5′ end of vip263 for R1 and the juxtaposed 3′end of the original vip269 and 5′ end of vip270 for R2, respectively.

Furthermore terminal PCR priming sites were introduced in the samereaction by the DNA ligase. The DNA oligoes having the PCR priming sitesvip029/vip070 and vip029/vip030 for R1 and R2, respectively, werepre-annealed under the following conditions: 200 pmoles of each oligo in1× Ligase buffer and 50 mM NaCl in total volume of 40 μl and incubatedin a PCR machine for 5 minutes at 95° C. and for 30 seconds steps at thefollowing temperatures: 80° C., 65° C., 50° C., 45° C., 30° C., 20° C.

When vip070 is annealed to vip029 the four most 5′ terminal nucleotidesof vip070 are protruding. These four are reverse complementary to thefour most 5′ terminal nucleotides in vip231 which also are protrudingwhen vip231 is annealed to vip263. Consequently, the protruding ends cananneal and form a substrate for a DNA ligase. Likewise, when vip030 isannealed to vip029 the four most 5′ terminal nucleotides of vip070 areprotruding. These four nucleotides are reverse complementary to the fournucleotides most 5′ in vip238 which also are protruding when vip238 isannealed to vip270. The cross-linked, gel-purified products werere-suspended in 1× Ligase buffer, 50 mM NaCl, and mixed with thepre-annealed PCR sites containing DNA oligoes; vip029/vip070 andvip029/vip030 for R1 and R2, respectively. The DNA 5′ ends were firstphosphorylated by 50 units Polynucleotide Kinase (NEB 0201L) in a finalvolume of 200 μl. The reaction was allowed to incubate for 30 minutes at37° C.

Then, the DNA was by a T4 DNA ligase transformed into a continuous DNAstrand consisting of vip029-vip231-vip262-vip263-vip070 andvip029-vip238-vip269-vip270-vip030 in R1 and R2 respectively. 1500 unitsT4 DNA Ligase (NEB 0201L) in 1× Ligase buffer, 50 mM NaCl were added tothe reactions giving a final reaction volume of 300 μl. The ligationreactions were incubated at 16° C. overnight. Then, the BSOCOES linkerwas eliminated as described above. The samples were precipitated byethanol and subsequently subjected to preparative non-native PAGE,ethanol precipitated again and dissolved in 20 μA water as describedabove. The assembled products were now ready for primer extension.

Throughout the above described procedure, small samples were removed foranalysis by none-native PAGE as described above. The gel picture isshown in FIG. 27. Lane 1 and 2 show the successful formed cross-linkedfunctionalised oligoes vip-231 (35 nt)/vip262 (68 nt) and cross-linkedvip-238 (35 nt)/vip-269 (68 nt) for R1 and R2, respectively. Thecross-linked products migrate with an apparent size of approximately 200bp. The observed difference between actually size and apparent size isnot unexpected due to the strong secondary structures in the formedproducts which are present even in a non-native gel due to the reversecomplementary sequences. Furthermore, bands of smaller size wereobserved which most likely originate from degradation of the full lengthspecie.

Lanes 3 and 4 show the products after both ligation of the two oligoesand elimination of the BSOCOES linker for R1 and R2 respectively. Themain products migrate with an apparent size of 150 bp. Furthermore,bands of smaller size were also observed most likely generated bydegradation of the full length species. Note that the species in bothlane 1 and 3 (and in lane 2 and 4) are of almost equal in size butmigrate significantly differently in the gel. This is not unexpectedbecause the species in lane 1 and 2 essentially are branched DNAmolecules, whereas the species in lane 3 and lane 4 are linear DNAmolecules.

Lane 5 and 6 show the product after cross-linking of the position 3oligoes for R1 and R2 respectively. In both reactions, the position 3oligo is 74 nt. Thus, the expected products are 177 nt. However, theapparent size in the gel of cross-linked species is around 600 bp. Thisis not unexpected because the species consist of cross-linked linear DNAmolecules with very strong secondary structures due to the reversecomplementary sequences in the arms of the DNA Star Structure. Even aurea containing PAGE are not capable of denature the structure.

Lane 7 and 8, contain the products after both ligation of the position 3oligoes and PCR sites containing oligoes and elimination of the BSOCOESlinker for R1 and R2 respectively. The PCR priming sites containingoligoes add a total of 64 nt, thus the desired products are 241 nt. Twoprominent bands of an apparent size in excess of 1000 bp were observed.The upper band most likely contains the full length products, whereasthe lower band most likely contains molecules missing one of the two PCRsites containing oligoes. Furthermore, in lane 8, a band migrating withan apparent size of 600 bp was seen. The band most likely represents aspecie with no PCR sites containing oligoes ligated (compare lane 8 and6).

Lane 9 contains the 100 bp DNA ladder (Fermentas, SM0248).

Primer Extension.

The assembled products were subjected to primer extension, whichtransforms the DNA folded in a star structure with the chemical productin the centre into linear double stranded molecules with the chemicalproduct displayed on the surface. The reaction was primed by vip038which is reverse complementary to the 3′ of the assembled molecules. Thereactions were conducted in 1× Thermopol buffer, 0.2 mM dNTPs, 8 mMMgSO4, 1 M betaine (Sigma, B-0300), 0.4 μM vip038, 0.2 U/10 μl Vent(exo-) (NEB M0259L), and 5 μl assemble molecule as template in a 10 μlprimer extension reaction. The reaction mix was incubated at 95□C for 2minutes, 60° C. for 30 seconds and at 74° C. for 5 minutes.

Binding Assay.

The DNA displaying the synthesized peptides was analyzed in aelectrophoresis mobility shift assay (EMSA). A demonstration of bindingwill confirm the capability of correctly synthesize a compound fromcarrier modules directed by Structural DNA.

This assay is adapted from the literature (Halpin and Harbury, PLoSBiol, 2, E174, 2004). 10 μL primer extension product of R1(Leu-enkephalin-DNA) or R2 (scrambled Leu-enkephalin-DNA) were eachplaced into 4 tubes. To tubes R1-1 and R2-1 1 μL 0.5 mg/ml 3E7 antiLeu-enkephalin monoclonal antibody (Chemicon, cat# MAB5276), 1 μL 1MTris-HCl pH 7.2 and 1 μL 0.1% Triton-X/PBS were added. To tubes R1-2 andR2-2 1 μL 0.5 mg/ml W6/32 monoclonal antibody (Sigma, H1650), 1 μL 1MTris-HCl pH 7.2 and 1 μL 0.1% Triton-X/PBS were added. To tubes R1-3 andR2-3 1 μL 0.5 mg/ml 3E7 anti Leu-enkephalin monoclonal antibody, 1 μL 20μM Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) (Schafer-N, Denmark) and 1 μL 1MTris-HCl pH 7.2 were added. To tubes R1-4 and R2-4 1 μL 0.5 mg/ml 3E7anti Leu-enkephalin monoclonal antibody, 1 μL 20 μM scrambledLeu-enkephalin (Gly-Leu-Phe-Tyr-Gly) (Schafer-N, Denmark) and 1 μL 1MTris-HCl pH 7.2 were added. All samples were incubated with agitationfor one hour at room temperature. 1.4 μL of 10× loading dye (Invitrogen,Cat#10816-015) was added to each sample and the entire amount was loadedonto the gel. 1 μL of both 100 bp and 1 kb ladders were also loaded(Fermentas #SM0248 and #SM0318). The 10% PAGE TBE gel (Invitrogen) wasrun cold at 220 mV and 15 mA for 45 minutes. The gel was developed for20 minutes in SYBR Green™ nucleic acid stain (Molecular Probes)according to manufactures instructions. The picture of the gel is shownin FIG. 28. Results: in all lanes two prominent bands having apparentsizes of 200-250 bp are observed. The bands most likely contain specieshaving double stranded DNA (further evidence can be found in e.g.Example 21). The upper band corresponds well with the intended 241 bpfull length product, whereas the lower band most likely contains aspecies missing the vip029 which will give a 30 bp smaller product. Twoprominent bands with apparent sizes of around 1000 bp are observed inall lanes. Most likely the bands contain species having star structurefolded DNA: the upper band most likely contains the full lengthproducts, whereas the lower band most likely contains molecules missingone of the two PCR sites containing oligoes.

In lane 1 containing R1-1 a band of apparent size of around 1500 bp isobserved. This band most contain the Leu-enkephalin-DNA product bindingto 3E7 antibody which slows the electrophoretic migration of the entirecomplex. This band is absent when the R1 product is incubated with theIgG2A isotype matched negative control antibody as shown in lane 2(R1-2). The specificity of the interaction is further enforced by thecompetition of the binding by free-soluble Leu-enkephalin: In lane 3 thegel-shifted band is absent when the R1 product, 3E7 and the freeLeu-enkephalin peptide are co-incubated. The competition is not seen inlane 4 containing R1-4 when the free soluble scrambled Leu-enkephalin ispresent. Lanes 5-8 containing R2-1, R2-2, R2-3 and R2-4, respectivelyshow that the negative control R2 product (scrambled Leu-enkephalin-DNA)does not bind to 3E7, as expected.

Amplification

To demonstrate that the DNA of the gel-shifted band and other bands areintact gel pieces were excised and DNA was extracted for use astemplates for PCR.

The gel pieces boxed in FIG. 29 were cut out and the product extractedby the “crush and soak” method (Sambrook, J., Fritsch, E F, andManiatis, T. (1989) in: Molecular Cloning: A Laboratory Manual, ColdSprings Harbor Laboratory); the gel piece was crushed and soakedovernight in 4000 μl TE Buffer. The tubes were spun for 10 minutes at 20000×g and the supernatant transferred to a fresh tube. PCR [1×Polymerase Buffer, 0.2 mM dNTP, 6 mM MgSO4, 0.2 μM vip027, 0.2 μMvip028, 0.5 M betain (Sigma, B-0300), 0.1 mg/ml BSA 0.08 units/μlVent(exo-)(NEB M0259L)] was then performed using 5 μl of the supernatantdiluted 200 fold in a 20 μl reaction. 20 cycles of 30″ at 95° C., 30″ at60° C. and 30″ at 74° C. in a thermocycler were performed and 2 μl ofthe samples were analyzed on a native 10% PAGE (Invitrogen) and stainedwith SYBR Green (Molecular Probes) according to manufacturesinstructions and a picture was taken. The result is shown in FIG. 29.Lane M contains the 100 bp DNA ladder (Fermentas, SM0248). Lanes 1-5contain the reactions having the gel purified templates: in all lanes aprominent band around 250 bp is observed corresponding to the expectedsize of the full length product. In contrast in lane 6 which contain thenegative control without template added the band is not observed.Consequently, it is hereby demonstrated that a Structural DNA Displayproduct can be formed, partitioned and amplified.

In conclusion, the specific binding of the R1 product to the 3E7anti-Leu-enkephalin antibody demonstrates conclusively that theLeu-enkephalin peptide has been correctly assembled by the process.Furthermore, it has been shown that partitioning of a product displayinga ligand from a product not displaying a ligand indeed is doable. Forexample as illustrated here simply by isolating the gel shifted band.Furthermore, the partitioned product can be amplified for subsequentlyidentification by e.g. DNA sequencing or used as a template in atranslation process, thus allowing cycles of selection and amplificationto be performed.

Example 19 DNA Star Structure Direction of Reductive Amination

The present example serves to illustrate that Structural DNA can directreductive amination. Reductive amination was chosen as an example of animportant and widely applicable chemoselective reaction.

Oligos were obtained from DNA Technology (Arhus, Denmark). Theoligonucleotide vip046 was acetylated with DST (disuccinimidyl tartrate,Pierce #20589) at the primary amine on an internal modified dT in theoligonucleotide followed by oxidative cleavage with NaIO₄ (Aldrich #31,144-8) to yield the glyoxylate functionalized oligo. The oligonucleotide(2.5 nmol) was treated with DST (10 mM) in a 40% DMF/water mixturecontaining and 400 mM pH 8.8 sodium phosphate buffer. Total volume ofthe reaction was 100 μL. The reactions were incubated 2 hrs at 25° C.NaIO₄ (50 μL of a 150 mM solution) was then added and incubated for anadditional 2 hrs at 25° C. The reaction mixture was diluted to 200 μLand purified on a spin column (Amersham Biosciences #27-5325-01)according to manufactures protocol followed by purification by HPLC andmass spectrometry analysis according to Example 11. Yield: 36%.

DNA Calculated mass Found mass vip046-NHCOCHO 6653.202 6656.1Synthesis of a Benzaldehyde-Functionalized Oligo Having InternalModified dT (Amine-C6-dT) (Position n=1) (Vip046-NHCOC₆H₄CHO)

The oligonucleotide vip046 was acylated with 4-carboxybenzaldehyde(Lancaster #8192) promoted by DMT-MM(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,Fluka #74104) by treatment of the oligonucleotide (2.5 nmol) dissolvedin a 40% DMF/water mixture containing 150 mM NaCl, 200 mM sodiumphosphate buffer pH 8.8 with DMT-MM 50 mM. Total reaction volume was 100μL. The reaction was incubated 4 hrs at 25° C. The reaction mixture waspurified on a spin column (Amersham Biosciences #27-5325-01) accordingto manufactures protocol followed by purification by HPLC and massspectrometry analysis according to Example 11. Yield: 53%

DNA Calculated mass Found mass vip046-NHCOC₆H₄CHO 6729.233 6729.9Structural DNA Directed Reductive Amination.

In reaction 1 and 5 the two derivatives of vip046 carrying an amide ofglyoxylic acid and 4-formylbenzoic acid, respectively, were mixed withequimolar amounts (10 pmoles each) of vip017 and vip008 in a buffersolution containing a final composition of morpholinopropanesulfonicacid (Fluka #69947; MOPS, 100 mM, pH 5.2) and NaCl (Fluka #71376, 1M).Solutions were subjected to an annealing program (PCR machine: 5 min @94° C., 30 sec @ 80° C., 30 sec @ 65° C., 30 sec @ 50° C., 30 sec @ 35°C., 30 sec @ 20° C., 30 sec @ 10° C.). The annealing mixture was addedthe reductant (NaCNBH₃; Sigma #156159, 1M aq. sol, final concentrationof 100 mM; total reaction volume 20 μL) and incubated for 2 h at 30° C.

Controls run in parallel for both aldehydes:

-   -   Vip017 was exchanged for vip007, which does not carry an amine        (reactions 2+6).    -   Vip008 was omitted in order to test the efficiency of the dimer        compared to trimer (reactions 3+7).    -   Vip046-CHO was attempted cross-linked to vip019, thus performing        the reaction between oligoes, which cannot base pair (reactions        4+8).

All eight reaction mixtures were diluted to 50 μl and EtOH precipitated(added GenElute 0.5 μL; Sigma 56575) and 96% EtOH (Biochemika grade; 250μl). Incubated 15 min on ice, then spun at 14000 rpm for 30 min at 4° C.Supernatant was decanted, tubes spun briefly, remaining liquid removedby pipette, and the pellet was allowed to dry in a stream of air).

Crude DNA was dissolved in water and analyzed by denaturing 10% PAGE(Invitrogen) and subsequently stained by SYBR Green (Molecular Probes)according to manufactures instructions. A picture of the gel is shown inFIG. 30. The formation of a new band with a mobility corresponding to60-70 nt confirms the expected cross-linking of vip046 (21 nt) andvip017 (42 nt) (lanes 5 and 9). No cross-linking was observed in thecontrol experiments with vip007, which is identical to vip017 exceptthat it's without an amine on the internal dT) indicating a selectivereaction (lanes 6 and 10). Same product was formed in the dimer, butslightly less intense bands were observed (lanes 7+11). No product wasobserved for oligoes that cannot base pair indicating that matchedsequences are required for product formation (lanes 8+12).

Consequently, Structural DNA is capable of directing reductive aminationin a highly specific manner.

Example 20 Structural DNA Direction of Urea Formation

The present example serves to illustrate that Structural DNA can directurea formation. Urea formation between two amines was chosen as anexample of an important, widely applicable reaction. Ureas are knownisosters in medical chemistry.

Oligos were obtained from DNA Technology Arhus, Denmark.

A DNA Star Structure consisting of a three-way DNA junction wasassembled comprising two amino functionalized oligoes and one auxiliaryoligo. Oligoes vip046, vip017, and vip008 were mixed in equimolaramounts (10 pmol each) in a buffer solution containing a finalcomposition of morpholinopropanesulfonic acid (Fluka #69947; MOPS, 100mM, pH 8.0) and NaCl (Fluka #71376, 1M). Solutions were subjected to anannealing program (PCR machine: 5 min @ 94° C., 30 sec @ 80° C., 30 sec@ 65° C., 30 sec @ 50° C., 30 sec @ 35° C., 30 sec @ 20° C., 30 sec @10° C.). The annealing mixture was urea forming reagents(N,N′-Disuccinimidyl carbonate, Aldrich #225827 (0.45M in DMF) orbis(4-nitrophenyl)carbonate, Aldrich #161691 (1.0M in DMF)) to a finalconcentration of 10, 50, or 100 mM (total reaction volume 20 μL) andincubated for 90 min at 37° C.

An aliquot of each reaction mixture was and analyzed by denaturing 10%PAGE (Invitrogen). Bands were visualized by SYBR Green stain (MolecularProbes, #S7563) according to manufactures instructions.

A picture of the PAGE is shown in FIG. 31. The formation of a new bandwith a mobility corresponding to 60-70 nt confirms the expectedcross-linking of vip046 (21 nt) and vip017 (42 nt) in lanes 5-10.Interestingly, a decreasing amount of product was formed with increasingamounts of coupling reagent. This observation, however, can be explainedby reactions of reagent molecules with both amino groups, thustransforming both amines into nucleophiles. Thus, a lower concentrationof reagent may allow for just one of the amines to form an intermediatecarbamate, which rapidly reacts with the other amine to form theexpected urea.

Consequently, Structural Star DNA is capable of directing ureaformation.

Example 21 DNA Star Structure Electromobility

This example demonstrates Structural DNA's electromobility in nativepolyacrylamide gels.

Structural DNA has a distinct different conformation than doublestranded DNA. The latter is a linear elongated molecule, whereasStructural DNA has a more globular structure. Consequently, differentmigration patterns of the two conformations are expected in gels:Structural DNA has an apparent size far exceeding that of the doublestranded linear DNA counterpart. To demonstrate this phenomenon thefollowing experiment was performed:

A trimeric DNA Star Structure with terminal PCR priming sites was formedby ligation of five oligoes vip029, vip 161, vip 192, vip 193 andvip207. A schematic drawing of the organization is shown in FIG. 32.

DNA oligoes (prepared by DNA Technology Århus, Denmark) were mixed in 2μM concentrations each in 1× Ligase Buffer (New England Biolabs), 50 mMNaCl. The mixtures were incubated as follows: 94 μC for 5 minutes, 80 μCfor 30 seconds, 65 μC for 30 seconds, 50 μC for 30 seconds, 35 μC for 30seconds, 20 μC for 30 seconds, 10° C. until next step. The annealingprocedure was performed on an Applied Biosystems AB2720 PCR machine. The5′ termini of the oligonucleotides were phosphorylated by T4 DNApolynucleotide kinase. A mixture consisting of 1.5 μM star structure,1×DNA ligase Buffer (New England Biolabs), 50 mM NaCl and 0.17 u/μl T4DNA polynucleotide kinase (New England Biolabs, cat# M0201), wasprepared and incubated for 30 minutes at 37° C. A phosphodiester bondbetween juxtaposed ends of annealed oligonucleotides was formed by T4DNA ligase (New England Biolabs, cat# M0202), in 1×DNA ligase Buffer(New England Biolabs), 50 mM NaCl, and 200 U T4 DNA ligase (New EnglandBiolabs, cat# M0202) in a volume of 10 p. 1 and incubated overnight at16° C.

PCR Amplification:

0.04 μl of the ligation reaction was used as template in 4000 PCRreaction mix [1× ThermoPol buffer (New England Biolabs B9004S), 0.2 mMdNTPs (New England Biolabs O447S), 8 mM MgSO4, 0.2 μM vip202 and vip224μM, 0.5 M Betaine (Sigma B0300), 1 U/100 μl of Vent (exo-) (New EnglandBiolabs M0257L). PCR amplification was performed in 50 μl aliquots usingthe following cycling conditions: 30 seconds at 92° C., and 25 cycles of92° C./15 sec, 50° C./15 sec, 70° C./30 sec.

The sample was ethanol precipitated by adding 1 ml ethanol and 1/10th 3M sodium acetate (pH 5.2), incubated 30 minutes on ice and centrifuged30 minutes at 20 000×g, the supernatant was discarded and the pellet wasresuspended in 1× loading buffer (Invitrogen) and subjected to apreparative native 10% PAGE (Invitrogen), and stained with SYBR Green(Molecular Probes, S7563) following manufactures instruction. A pictureof the gel is shown in FIG. 32. Lane M contains the 100 bp DNA ladder(Fermentas, SM0248). Two bands were isolated: the band around 250 bp (A)and the band with an apparent size in excess of 1000 bp. The gel piecesboxed in FIG. 32 were cut out and the product extracted by the “crushand soak” method (Sambrook, J., Fritsch, E F, and Maniatis, T. (1989)in: Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory); the gel piece was crushed and soaked overnight in 400 μl TEBuffer. The tubes were spun for 10 minutes at 20 000×g and thesupernatant transferred to a fresh tube and ethanol precipitated asdescribed above and resuspended in 100 μl water.

Primer Extesion

Primer extensions using the isolated DNA as templates was thenperformed. 2 μl template was used in a 20 μl reaction containing [1×ThermoPol buffer (New England Biolabs B9004S), 0.2 mM dNTPs (New EnglandBiolabs O447S), 8 mM MgSO4, 0.5 M Betaine (Sigma B0300), 1 U/100 μl ofVent (exo-) (New England Biolabs M0257L)]. To reactions 1 and 5 noprimer was included, in reactions 2 and 6 0.2 μM vip202 were included,in reaction 3 and 7 0.2 μM vip224 were included and in reactions 4 and 80.2 μM vip202 and 224 were included. The primer extensions wereperformed by incubating the samples at 95□C for one minute, at 50□C for15 seconds and at 70□C for 30 seconds. 10 μl of the reactions wereanalyzed by 10% PAGE as described above.

Discussion and Conclusion

First, PCR was performed using a trimeric DNA Star Structure withterminal PCR priming sites as template. Secondly, the sample wassubjected to preparative PAGE where two bands were isolated: a band (A)with an apparent size of around 250 bp (the expected size of the doublestranded PCR product is 241 bp) and a band (B) with an apparent size inexcess of 1000 bp. Finally, the isolated DNA was used as templates inprimer extension reactions and analyzed by PAGE. As shown in FIG. 32,both templates give rise to a double stranded product with a size ofaround 250 bp, with both the forward (lanes 2 and 6) and the backwardprimer (lanes 3 and 7). Furthermore, when both primers are present moreproduct is formed (lanes 4 and 8). This is illustrating that both band Aand B contain the intended 241 bp product and the difference in mobilityin the gel are consequently due to folding: A is presumably the doublestranded linear product, whereas the B is Star Structure folded DNA.

Interestingly, without any primers present in the primer extensionreaction only a limited double stranded band around 250 bp is observednot even when the starting was double stranded DNA as shown in lane 1.This is properly due to the thermal de-naturation and renaturation cyclethe sample has undergone, which will lead to the formation of a mixedproduct of double stranded DNA and Star Structure folded DNA. The samephenomenon is observed in lane 5 where the starting material is band B.

Consequently, it is hereby demonstrated that the 241 nucleotide long DNAmolecule can fold into a conformation (Star Structure), which leads to aapparent size in a native gel in the excess of 1000 bp.

Example 22 Translation of DNA Star Structures

This example demonstrates the principle of the translation process ofDNA Star Structures. In this context the translation process is theprocess where the individual modules on various positions aresubstituted by fresh modules and the substitution process is directed bycodon/anticodon recognition. The fresh modules may have a chemicalreactant attached in such a way that it will be located in the centre orin the vicinity of the centre upon proper folding of the new DNA StarStructure which the fresh modules will be a part of. Consequently,translation allows the chemical compound encoded by the DNA StarStructure to be synthesized.

The starting material for the translation process may be a PCR productusing the output from a selection process as a template for example asshown in Example 18. This will allow iterative cycles of selection andamplification, which in turn will allow a diverse library to converttowards solutions for the applied selection pressure.

Outline of the Translation Process

A schematic drawing of the major steps of a translation process using aPCR product as starting material are shown in FIG. 33.

First, a DNA Star Structure was amplified by PCR using a biotinylatedbackward primer, which allowed the separation of the two strands. Theseparation was performed by using magnetic streptavidin beads. Thestrand of interest (upper strand) was eluted from the beads and foldedinto the DNA Star Structure having two stem-loops and stem. Then, theposition 1 stem (without a loop) was digested by the restriction enzymeBsa I, which digest outside its recognition sequence and formed a 5′overhang. This overhang was the codon for position 1.

Then a fresh carrier module for position 1 having a suitable 5′anticodon sequence was ligated to the Star Structure. To aid theligation and downstream purification a biotinylated helper oligo wasincluded. The helper oligo hybridize to the fresh position 1 carriermodule in such a way that it created a 5′ overhang, which was theanticodon. Consequently, the subsequent ligation (helper oligo/StarStructure/module 1 was guided by the codon/anticodon hybridization.

Then, the original module on position 1 was liberated from the StarStructure by first performing a denaturation step where the freshcarrier module was replacing the original position 1 module in the StarStructure and then a restriction enzyme digest was performed in thesequence which was originally located in the distal loop in the secondstem. By this exercise the covalent bond between the original position 1module as well as the base pairing to the Star Structure were removed.The restriction enzyme digest was performed on Star Structure capturedon streptavidin coated magnetic beads. Consequently, Star Structuresliberated from the beads were successfully translated for postion 1.

Note that upon folding to the Star Structure, the 3′ end of the freshposition 1 module was participating in forming the second stem and thestem ends immediately before the codon on position 2. Consequently the3′ end of the fresh module 1 was lined up for accepting a fresh carriermodule for position 2 directed by codon/anticodon interactions forposition 2. Consequently, the Structure was ready for the secondcodon/anticodon directed module substitution. Consequently, by repeatingthe described substitution process for all positions in the StarStructure a complete translation is accomplished.

Star Structure Formation

The first step involved annealing of five oligoes: vip206, vip161,vip192 and vip193 vip207. A schematic drawing of the organization isshown in FIG. 34A. The five oligoes in the reaction will hybridize toeach other, thus forming a three-way junction, consisting of twostem-loops and one stem with both 5′ and 3′ un-hybridized sequences atthe end distal to the centre of the three-way junction. Theun-hybridized sequences represent PCR priming sequences (5′ segment ofvip206 and 3′ segment of vip207). The oligoes were mixed in 1× Ligasebuffer (NEB B0202S), 50 mM NaCl, with 20 pmol of each oligonucleotide ina volume of 10 μl. The annealing was performed by incubation in a PCRmachine for 5 minutes at 95° C. and for 30 seconds steps at thefollowing temperatures: 80° C., 65° C., 50° C., 45° C., 30° C., 20° C.

Then, the 5′ ends were phosphorylated by Polynucleotide Kinase; 2.5units Polynucleotide Kinase (NEB M0201) were included in a 15 μlreaction volume, in 1× Ligase Buffer, 50 mM NaCl. The reaction wasincubated for 30 minutes at 37° C.

The annealed oligoes were transformed into a continuous DNA strand by aDNA ligase, which formed phosphordiesther bonds between the juxtaposed3′ end of vip206 and the 5′ end of vip161, and between the juxtaposed 3′end of vip161 and the 5′ end of vip192, and between the juxtaposed 3′end of vip192 and the 5′ end of vip 193, and between the 3′ end of vip193 and the 5′ end of vip207, respectively. T4 DNA ligase (NEB M0202L)was added in 1× Ligase Buffer, 50 mM NaCl, and 20 units/μl enzyme wereadded, giving a final reaction volume of 20 μl. The ligation wasincubated overnight at 16° C. The DNA Star Structure was PCR amplifiedin a total reaction volume of 400 μl, in 1× Thermopol Buffer (NEBM0257L), 8 mM MgCl₂, 0.2 mM dNTPs, 0.5 M Betaine (Sigma B0300), 1 μg/mlBSA (NEB B9001S), 0.1 μM primers (vip202 and vip224) and 32 unitsVent(exo-) (NEB M257L). Vip224 had a biotin moiety at the 5′ end,enabling capture of the PCR product on streptavidin coated magneticbeads. Amplification was performed with an initial denaturation step for30 seconds at 92° C., followed by 25 cycles with incubations at 92° C.for 30 seconds, at 60° C. for 15 seconds and at 70° C. for 30 seconds. Afinal extension at 70° C. for 1 minute was done.

After the PCR amplification, the PCR products were cleaned up using theEppendorf kit (0032 007.740) according to the instructions. Two columnswere used. Elution was done with 150 μl TE for each column. Then, thecleaned up PCR product was added to Streptavidin coated magnetic beads(Dynal MyOne, 605.02). 100 μl beads were washed two times in 2×BWTBuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 M NaCl, 0.1% Triton X-100).The beads were suspended in 300 μl 2×BWT, and 300 μl cleaned up PCRproduct was added to the beads. The beads were incubated for 15 minutesat RT, shaking slowly. The beads were captured on a magnet, and thesupernatant was removed. The beads were washed 3 times with 1×BWT (halfstrength of 2×BWT). The DNA was eluted from the complementarybiotinylated DNA strand captured on the magnetic beads by adding 50 μl10 mM NaOH, and incubated for 5 minutes at room temperature. The beadswere captured on the magnet, the supernatant containing the upper DNAstrand was removed and immediately neutralized with 20 μl 1 M Tris-HCl,pH 7.2.

The purity of the eluted DNA was tested by primer extension reactionswith the forward and reverse primers in separate reaction. The primerextension reactions was performed using Vent(exo-) (NEB), in reactionsof 10 μl, in 1× Thermopol Buffer, 8 mM MgCl₂, 0.2 mM dNTPs, 1 M Betaineand 0.2 units Vent(exo-) and 0.2 μl template and 0.4 μM vip202 or 0.4 μMvip203 per reaction. The primer extension reaction was as follows: 95°C. for 2 minutes, 60° C. for 30 seconds, and 74° C. for 5 minutes.

An outline of the procedure is shown in FIG. 34A. Through out theprocedure samples were removed for analysis by PAGE. A picture of thegel is shown in FIG. 34B. The gel also contains the quality controlprimer extension reactions. In lane 2 is observed a band of the expectedsize of 241 bp, which is not present in the negative control withoutadded template (lane 1). Consequently, the DNA Star Structure has beensuccessfully assembled and amplified. Furthermore, two bands with anapparent size in the excess of 1000 bp are observed in lane 2, whichrepresents folded DNA Star Structure (for reference see Example 21).Lane 3 shows the product after PCR clean up.

The product was captured via the biotin moiety on the lower strand ofthe PCR product. The flow through is shown in lane 4. The lower of thetwo bands with an apparent size in the excess of 1000 bp was found here,which consequently did neither contain biotin nor was hybridized to thebiotinylated strand. After washing of the beads, the unbiotinylatedstrand was eluted by high pH which abolished base pairing. The elutionproduct is shown in lane 5. A single band equivalent to the lower of thetwo bands with an apparent size in the excess of 1000 bp was observedindication successful isolation of the un-biotinylated upper strand ofthe PCR product. Interestingly, the upper band of the two bands with anapparent size in the excess of 1000 bp was only observed when bothstrands of the PCR were present (Lanes 1 and 2). Consequently, the upperband most likely represents two hybridized DNA Star Structuremolecules—hybridized via the terminal PCR priming sites.

As a quality control of the purified DNA two primer extensions wereperformed. In one reaction the forward primer (vip202) was used and inthe second reaction the backward primer (vip203) was used, which primeon the lower and upper PCR strands, respectively. Consequently, if theisolated DNA is pure only the second reaction should give rise to aprimer extension product. Accordingly, only a band corresponding to theexpected size of 241 bp was observed in lane 7 whereas no primerextension product was observed in lane 6. Consequently, the successfulpurification of the desired strand of the PCR product in a highly purepreparation was achieved.

Exposure of the Codon on Position I

The DNA Star Structure contained two stem-loops and one stem. The stemwithout a loop contained the codon for position 1. The codon was exposedby restriction enzyme digest by Bsa I, which cut outside its recognitionsequences and formed a 4 nucleotide 5′ overhang, which sequence can bechosen without restrictions, thus ideal for encoding purposes. In thiscontext the overhang is called the codon for position 1.

The Star Structure DNA was subjected to digest with Bsa I. The doublestranded substrate for Bsa I was found in the first stem (the stemwithout a loop) generated by hybridization of the 5′ segment of vip 161and the 3′ segments of vip 193. Note that the product obtained after theBsaI digest corresponds to the sequence of vip 161-vip 192-vip193described at the start of this example.

110 μl purified Star Structure DNA were mixed with 20 μl 10×NEB3 bufferand 200 units of Bsa I (NEB R0535L) in a total volume of 200 μl. Thedigest was incubated at 50° C. for 2.5 hours. The DNA was subjected to astandard ethanol precipitation, before it was applied to a 10% TBE-ureagel (Invitrogen) for gel purification.

In FIG. 35 is shown a picture of the gel after SYBR green staining.Uncut DNA was loaded in lane 1 as a reference. A prominent bandmigrating with an apparent size in the excess of 1000 bp is observed(Note the “spil over” from the marker loaded in lane M). Multible bandswere observed in lanes 2-7 where the Bsa I digest was loaded, thusindicating that the BsaI digest was not complete. However, the band ofinterest was excised from the gel (boxed on the figure), and the DNA wasextracted from the gel piece by the “crush and soak” method, ethanolprecipitated and redissolved in 40 μl H₂O as described previously.

Ligation of Fresh Position 1 Module Directed by Codon/AnticodonInteraction.

Fresh position 1 module, vip271, was ligated onto the Bsa I digested andpurified DNA Star Structure. Vip066 was included in the ligationreaction as a ligation aid, and to introduce a biotin molecule into theligation product, thus facilitation downstream purification. Annealingof vip271 and vip066 will generate a product with a 4 nucleotide 5′overhang (vip271) which in this context is called the anticodon. Thesequence was therefore chosen in such a way that it was reversecomplement to the codon on position 1 in the DNA Star Structure.Consequently codon/anticodon hybridization is capable of guiding theligation of the two incoming oligoes with the DNA Star Structure. Vip271and vip066 were mixed in 1× Ligase buffer (NEB B0202S), 50 mM NaCl, with50 pmoles of each oligonucleotide in a volume of 10 μl. The annealingwas performed on a PCR machine using the annealing program describedabove.

Then, the annealed vip271/vip066 were mixed with the Bsa I digested andpurified DNA Star Structure (30 μl), and the 5′ ends were phosphorylatedby Polynucleotide Kinase; 12.5 units Polynucleotide Kinase (NEB M0201)were included in a 50 μl reaction volume in 1× Ligase Buffer, 50 mMNaCl. The reaction was incubated for 30 minutes at 37° C.

Then, the cognate ends of the molecules were joined by a DNA ligase,which formed a phosphordiesther bond between the juxtaposed 3′ end ofvip066 and 5′ end of the Bsa digested DNA, and between the juxtaposed 3′end of the BsaI digested DNA 5′ end of vip271. T4 DNA ligase (NEBM0202L) was added in 1× Ligase Buffer, 50 mM NaCl, and 20 units/μlenzyme were added, giving a final reaction volume of 60 μl. The ligationwas incubated overnight at 16° C.

Substitution

The next step was to eliminate the original position 1 module from theStar Structure. The molecule was re-folded allowing the fresh position 1module to be part of the three-way junction and a covalent bond betweenthe original position 1 and the DNA Star Structure was eliminated.Furthermore, a helper oligo (vip194) was introduced, which did anneal tothe Pvu. II site in the original loop in the distal end of the 2^(nd)stem (see FIG. 36) thus forming a double stranded substrate for Pvu II.Consequently, both the covalent bond and the base pairing were destroyedbetween the original position 1 module and the Star Structure.Furthermore the fresh position 1 module was introduced as a part of thethree-way junction.

43 μl of the ligation reaction was mixed with 200 moles of vip194 in 10mM Tris-HCl pH 8, 1 mM EDTA, 100 mM NaCl, 0.1% Triton X-100 in a totalvolume of 60 μl. The denaturation and annealing was performed in the PCRmachine for 5 minutes at 95° C. and for 30 seconds steps at thefollowing temperatures: 80° C., 65° C., 50° C., 45° C., 30° C., 20° C.

The DNA was then captured on Streptavidin-coated magnetic beads (Dynal).30 μl beads were washed 2× in 2×BWT (2 M NaCl, 10 mM Tris-HCl, pH 8, 1mM EDTA, 0.1% Triton X-100). After the final wash, the beads weresuspended in 60 μl 2×BWT, and 60 vip194/Star Structure annealingreaction was added. Incubation was done for 15 minutes at roomtemperature with gentle shaking. The beads now with the DNA attached tothem, were captured on a magnet and subsequently suspended in Pvu IIdigest Buffer: 22 μl H₂O and 3 μl 10× Pvu II digest Buffer was added tothe beads, and 5 μl Pvu II (10 units/μl; Fermentas ER0637) were added.The digest was incubated for 6 h at 37° C. After the digest, the beadswere separated from the supernatant on the magnet.

Throughout the procedure aliquots was saved for analysis by 10% TBE-ureagel (Invitrogen) stained with SYBR green (Molecular Probes) according tothe manufactures protocol. A picture of the gel is shown in FIG. 36.

The purified Bsa I digested Star Structure was loaded in lane 1; aprominent band of the expected apparent size of around 600 nt wasobserved. The ligation product of the Bsa I digested Star Structure withfresh module for position 1/helper oligo (vip066) was loaded in lane 2.A prominent band with an apparent size in the excess of 1000 nt wasobserved, thus indicating a successful ligation. The beads after the PvuII digest were loaded in lane 4. A band with an apparent size in theexcess of 1000 nt was observed. This band corresponds to undigested DNAwhich is seen by comparison to lane 2. However, in the supernatant fromthe Pvu II digest (lane 5) a band with an apparent size of around 600 ntwas observed. This band corresponds to the expected apparent size ofaround 600 nt of a successful substitution product. The fact that it wasfound in the supernatant show that the fresh module on position 1 hassubstituted the original module on position 1 in the star structure.

Consequently, successful translation, i.e. codon/anticodon directedmodule substitution has been demonstrated.

List of oligonuclotides oligonucleotides used in the examples

Name SEQ ID NO: Sequence Modification vip006 SEQ ID NO: 14CTCGTTTTCGAGACCGACTCTGGAAGTGTCACCGGATCTGG 5′P vip007 SEQ ID NO: 15TTGGAAAAACCAACCAGATCCGGTGACTGTCAAGGCTGAGGT 5′P vip008 SEQ ID NO: 16GAGGGAGAGCCTCACCTCAGCCTTGACTCTTCCAGAGTCGGT 5′P vip009 SEQ ID NO: 17GAGGGAGAGCCTCACCTCAGCCTTGACTGGAGAACGCATTCT 5′P vip010 SEQ ID NO: 18ACACAAGAAGTGTAGAATGCGTTCTCCTCTTCCAGAGTCGGT 5′P vip016 SEQ ID NO: 19CTCGTTTTCGAGACCGACTCTGGAAGXGTCACCGGATCTGG X = amine-C6-dT vip017SEQ ID NO: 20 TTGGAAAAACCAACCAGATCCGGTGACXGTCAAGGCTGAGGT X = amine-C6-dTvip018 SEQ ID NO: 21 GAGGGAGAGCCTCACCTCAGCCTTGACXCTTCCAGAGTCGGT X =amine-C6-dT vip019 SEQ ID NO: 22GAGGGAGAGCCTCACCTCAGCCTTGACXGGAGAACGCATTCT X = amine-C6-dT vip020SEQ ID NO: 23 ACACAAGAAGTGTAGAATGCGTTCTCCXCTTCCAGAGTCGGT X = amine-C6-dTvip027 SEQ ID NO: 24 ACTATGAGGGCTGTCTGTGG None vip028 SEQ ID NO: 25TAGCAAGCCCAATAGGAACC None vip029 SEQ ID NO: 26ACTATGAGGGCTGTCTGTGGCAGTCACGAG None vip030 SEQ ID NO: 27AAAACTCGTGACTGGGTTCCTATTGGGCTTGCTA 5′P vip031 SEQ ID NO: 28TTTTCGAGACCGACTCTGGAAGTGTCACCGGATCTGG 5′P vip034 SEQ ID NO: 29ACTATGAGGGCTGTCTGTGG 5′ biotin vip038 SEQ ID NO: 30 TAGCAAGCCCAATAGGAACC5′ biotin vip046 SEQ ID NO: 31 ACTCTGGAAGXGTCACCGGAT X = amine-C6-dTvip048 SEQ ID NO: 32 GAGGGAGAGCCTCACCTCAGCCTTGACACACACXCTTCCAGA 5′P, X =amine- GTCGGT C6-dT vip056 SEQ ID NO: 33CTCGTTTTCGAGACCGACTCTGGAAGAGTGTGTTGTCACCGGA TCTGG vip068 SEQ ID NO: 34CAGCCTTGACXCTTCCAGAGT X = amine-C6-dT vip070 SEQ ID NO: 35CTCTCTCGTGACTGGGTTCCTATTGGGCTTGCTA vip076 SEQ ID NO: 36ACTCTGGAAGXGTCACCGGATCTGG X = amine-C6-dT vip078 SEQ ID NO: 37GAGGGAGAGCCTCACCTCAGCCTTGACTCTTCCAGAGTGGTT CCTATTGGGCTTGCTA vip132SEQ ID NO: 38 TTGGAAAAACCAACCAGATCCGGTGACTGTGTGTGTCAAGG CTGAGGT vip133SEQ ID NO: 39 GAGGGAGAGCCTCACCTCAGCCTTGACACACACTCTTCCAG AGTCGGTCTCGvip161 SEQ ID NO: 40 AGAGCGAGACCGACTCTGGAAGTGTCACCGGATCT vip162SEQ ID NO: 41 GGTTGGCAGGGCCCACTAGCTCAGGATCCACCCAACCAGATCCGGTGACTGTGTGTGTCAAGGCTGAG vip163 SEQ ID NO: 42GTGAGGCTGAATTCTCTGTACCTGGTACCTCCCTCACCTCAGCCTTGACACACACTCTTCCAGAGTCGGTCTCG vip164 SEQ ID NO: 43 GTGGGCCCTG vip165SEQ ID NO: 44 GTGGATCCTG vip192 SEQ ID NO: 45GGTTGGCACAGCTGACTAGCTCAGAGCTCACCCAACCAGAT CCGGTGACTGTGTGTGTCAAGGCTGAGvip193 SEQ ID NO: 46 GTGAGGCTCCCGGGTCTGTACCTATTAATTCCCTCACCTCAGCCTTGACACACACTCTTCCAGAGTCGGTCTCG vip194 SEQ ID NO: 47 GTCAGCTGTG vip195SEQ ID NO: 48 GTGAGCTCTG vip066 SEQ ID NO: 49 XCTTCCAGAGTCGGTCTCG X = 5′bio vip088 SEQ ID NO: 97 XCAGCCTTGACTCTTCCAGAGT X = 5′ bio vip202SEQ ID NO: 98 CAGGTCGCTGAGAGGTTGAC vip203 SEQ ID NO: 99ACGTCCGAGTCAGAAGTGTG vip206 SEQ ID NO: 100CAGGTCGCTGAGAGGTTGACCAGTCACGAG vip207 SEQ ID NO: 101CTCTcTCGTGACTGCACACTTCTGACTCGGACGT vip224 SEQ ID NO: 102XACGTCCGAGTCAGAAGTGTG X = 5′ bio vip231 SEQ ID NO; 103AGAGCGAGACCGACTCTGGAAGXGTCACCGGATCT X = NH2-C6-dT vip238 SEQ ID NO: 104TTTTCGAGACCGACTCTGGAAGXGTCACCGGATCT X = NH2-C6-dT vip262 SEQ ID NO: 105GGTTGGCACAGCTGACTAGCTCAGAGCTCACCCAACCAGAT X = NH2-C6-dTCCGGTGACTGTGTGXGTCAAGGCTGAG vip263 SEQ ID NO: 106GTGAGGCTCCCGGGTCTGTACCTATTAATTCCCTCACCTCAG X = NH2-C6-dTCCTTGACACACACXCTTCCAGAGTCGGTCTCG vip269 SEQ ID NO: 107GGTTGGCACAGCTGACAAAAACAGAGCTCACCCAACCAGAT X = NH2-C6-dTCCGGTGACTGTGTGXGTCAAGGCTGAG vip270 SEQ ID NO: 108GTGAGGCTCCCGGGTCGAGAGCTATTAATTCCCTCACCTCAGC X = NH2-C6-dTCTTGACACACACXCTTCCAGAGTCGGTCTCG vip271 SEQ ID NO: 109CTCTCGAGACCGACTCTGGAAGXGTCACCGGATCTGGTTGGGT- X = NH2-C6-dT GAGCTCTG

The invention claimed is:
 1. A method for creating one or more chemical compounds comprising the steps of: (i) providing N (N=3-100) carrier modules comprising: (1) a first position carrier module having a) a nucleic acid segment capable of hybridizing to a nucleic acid segment of the N position carrier module, and b) a nucleic acid segment capable of hybridizing to a segment of a second position carrier module, (2) n position carrier module(s) (occupying positions 2 to N−1) having a nucleic acid segment capable of hybridizing to a nucleic acid segment of a n−1 carrier module, and a nucleic acid segment capable of hybridizing to a segment of a n+1 carrier module, and (3) a N position carrier module having a nucleic acid segment capable of hybridizing to a nucleic acid segment of the N−1 carrier module, and a nucleic acid segment capable of hybridizing to a segment of said first carrier module, wherein at least three of said carrier modules comprise an associated chemical group (CG) situated in the mid section between the hybridization segments and optionally a codon segment situated external to one of the hybridization segments; wherein said mid section comprises 1 to 20 nucleotides, and each hybridization segment consists of 12 or more nucleotides; (ii) contacting said carrier modules under conditions allowing hybridization of said hybridization segments so as to form a structure resembling a star, wherein said star structure comprises a reaction center and a plurality of stems, and each stem is formed by two hybridizing segments complementing each other, and at least one of the stems extends radially from the reaction center so that the mid section between the two hybridization segments on each carrier module is pointing towards the center, thus bringing said chemical groups into reactive proximity; and (iii) providing conditions allowing chemical reaction(s) of the chemical groups resulting in the formation of a chemical compound, where the chemical compound is covalently associated with at least one of said carrier modules.
 2. A method according to claim 1 further comprising the step of providing conditions allowing ligation of the termini of module n−1 to module n and module N−1 to module N, thereby forming a continuous nucleic acid molecule with stem-loop structures and a chemical compound associated therewith.
 3. The method according to claim 2, wherein said ligations are performed enzymatically or chemically; and sequentially, or simultaneously.
 4. The method of claim 1, wherein the chemical compound is covalently associated with at least one of said carrier molecules or a continuous nucleic acid molecule resulting from ligation of said carrier modules.
 5. The method according to claim 1, wherein the contacting of the carrier modules is performed sequentially or simultaneously.
 6. The method according to claim 1, wherein the chemical reaction(s) are performed sequentially or simultaneously.
 7. The method according to claim 1, further comprising a priming site for a DNA polymerase, RNA polymerase or reverse transcriptase site in at least the first carrier module and/or at least in the N carrier module.
 8. The method according to claim 1, comprising the further step of performing an enzymatic extension reaction to display the chemical compound.
 9. The method according to claim 1, wherein N is 3, 4, 5, 6 or 7 and wherein each of the carrier modules comprise an associated chemical group (CG) situated in the mid section and a codon segment situated external to one of the hybridization segments.
 10. The method according to claim 1, wherein a library of more than one compound is synthesized by having a repertoire of carrier modules on one or more positions.
 11. The method according to claim 10, wherein the repertoire on at least one position comprises at least 10 different carrier modules.
 12. The method according to claim 10, wherein the repertoire on at least two positions comprises at least 10 different carrier modules.
 13. The method according to claim 1, wherein the chemical group is associated with a nucleobase of the mid section.
 14. The method according to claim 1, wherein the chemical group is associated with a phosphodiester linkage of the midsection.
 15. The method according to claim 1, wherein the chemical group is associated to the midsection through one or more covalent bonds.
 16. A method for screening a library of more than one chemical compound prepared as disclosed in claim 8, comprising the steps of: probing the library for library members having a chemical compound of desired property; partitioning the library members having desired property from library members not having desired property; and thereby obtaining an enriched pool of library members having desired property.
 17. The method according to claim 16, wherein the method further comprises amplifying the nucleic acid of the members of the enriched pool, said nucleic acid being indicative of the history of the chemical reaction(s). 